BETTER MICROSCOPY

A Series of Practical User’s Guides

Volume I

Guide to the “Secrets” of

Transmitted-Light Microscopy

D. J. Jackson

Copyright by David J. Jackson, 2008. All rights reserved.

Revised Edition
 Contents
Chapter Page
--- Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-v

1.0 An Introduction to “Real World” Microscopy . . . . . . . . . . . . I-1

1.1 A “Real World” View of Microscopy . . . . . . . . . . . . . . . . I-1
1.2 Some Popular Misconceptions . . . . . . . . . . . . . . . . . . . . . I-2
1.3 “Secrets” of Good Microscopy . . . . . . . . . . . . . . . . . . . . . I-2
1.4 Lab vs. Stereo Microscopes . . . . . . . . . . . . . . . . . . . . . I-3
1.5 Lab Microscope Types . . . . . . . . . . . . . . . . . . . . . . . . . . . I-4
1.6 Some Things to Avoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-8
1.7 Research and Vintage Microscopes . . . . . . . . . . . . . . . . . . I-10
1.8 What Defines a “Good” Microscope? . . . . . . . . . . . . . . . . I-13
1.9 Pursuit of the Perfect Image . . . . . . . . . . . . . . . . . . . . . . . . I-14
1.10 “Optimizing” your Scope! . . . . . . . . . . . . . . . . . . . . . . . . . I-17

2.0 “Real World” Microscope Optics . . . . . . . . . . . . . . . . . . . . . . . I-23

2.1 “Real World” Optics -- a Demonstration . . . . . . . . . . . . . . I-23
2.2 Optics and the Microscope . . . . . . . . . . . . . . . . . . . . . . . . I-24
2.3 Microscope Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . I-25
2.4 Microscope Eyepieces . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-28
2.5 Image Formation -- Critical Factors . . . . . . . . . . . . . . . . . I-31
2.6 The “Mix-n-Match” Game . . . . . . . . . . . . . . . . . . . . . . . . . I-35
2.7 The Great “170 vs 160mm” Debate . . . . . . . . . . . . . . . . . . I-38
2.8 Parfocal Distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-39
2.9 “Infinity” Optical Systems . . . . . . . . . . . . . . . . . . . . . . . . . I-41
2.10 “Real World” Choices . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-43

(cont’d . . . )

I-i
 Contents (cont’d)
Chapter Page
3.0 Microscope Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-45
3.1 “Real World” Microscope Illumination . . . . . . . . . . . . . . . I-45
3.2 Illumination and the Condenser . . . . . . . . . . . . . . . . . . . . . I-47
3.3 Kohler vs. Critical Illumination . . . . . . . . . . . . . . . . . . . . . I-48
3.4 “Simplified” Kohler Illumination . . . . . . . . . . . . . . . . . . . . I-50
3.5 Higher-Performance Condensers . . . . . . . . . . . . . . . . . . . . I-52
3.7 Proper Illumination? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-56
3.8 “Verifying” Your Illumination . . . . . . . . . . . . . . . . . . . . . . I-58
3.9 Correcting Illumination Faults . . . . . . . . . . . . . . . . . . . . . . I-60
3.10 Illumination and the “Real World” . . . . . . . . . . . . . . . . . . I-63
3.11 Battle of the Irises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-65
3.12 Kohler and Glare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-69

4.0 “Real World” Microscope Use . . . . . . . . . . . . . . . . . . . . . . . . . I-73

4.1 The Compound Microscope . . . . . . . . . . . . . . . . . . . . . . . . I-73
4.2 Basic Illumination Setup . . . . . . . . . . . . . . . . . . . . . . . . . . I-74
4.3 The Ubiquitous Abbe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-74
4.4 The Binocular Body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-75
4.5 Binocular Adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-77
4.6 Low-Power Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . I-78
4.7 Medium Power Observation . . . . . . . . . . . . . . . . . . . . . . . . I-80
4.8 High-Power Observation . . . . . . . . . . . . . . . . . . . . . . . . . . I-82
4.9 The Oil Immersion Objective . . . . . . . . . . . . . . . . . . . . . . . I-84
4.10 Using Oil Immersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-85

Appendix I – Glossary of Microscopy Terms . . . . . . . . . . . . . . . . . . . . . . . . I-89

Appendix II – Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-93
Appendix III – Internet-based Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-95
Combined Index (for Vols. I & II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II-41

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 List of Tables
Section Table or Summary Page
1.4 Important Microscope Features . . . . . . . . . . . I-3
1.5 Lab Microscope Types . . . . . . . . . . . . . . . . . . . I-4
Differences Between Types . . . . . . . . . . . . . . . I-5
Basic Features and Functions . . . . . . . . . . . . . . I-7
1.6 Signs of Cost-Cutting . . . . . . . . . . . . . . . . . . . . I-8
Consistent Weaknesses . . . . . . . . . . . . . . . . . . I-9
1.7 Research Microscope Summary . . . . . . . . . . . . I-10
Vintage Microscope Summary . . . . . . . . . . . . . I-11
1.8 Important Microscope Features . . . . . . . . . . . . I-13
1.9 Objective Resolution Limits . . . . . . . . . . . . . . I-15
Common Microscope Limitations . . . . . . . . . . I-15
2.3 Common Objective Parameters . . . . . . . . . . . . I-26
“Critical” Objective Parameters . . . . . . . . . . . . I-27
2.4 Common Eyepiece Characteristics . . . . . . . . . . I-28
Typical Image Color Faults . . . . . . . . . . . . . . . I-29
2.5 Common Refractive Index Values . . . . . . . . . . I-32
Sensitivity to Coverglass Thickness . . . . . . . . . I-33
2.6 “Mix-n-Match” Summary . . . . . . . . . . . . . . . . I-35
2.8 Typical Parfocal Distances . . . . . . . . . . . . . . . . I-39
3.3 Strict Kohler illumination Conditions . . . . . . . I-48
Setup steps for Kohler Illumination . . . . . . . . . I-49
Simplified Kohler Setup Procedure . . . . . . . . . I-49
3.4 Kohler vs. Critical Illumination . . . . . . . . . . . . I-51
3.5 Condenser Types and Characteristics . . . . . . . I-53
3.6 Special-Purpose Condensers . . . . . . . . . . . . . . I-54
Abbe Condenser “Adaptations” . . . . . . . . . . . . I-55
3.9 “Illumination Viewing” Alternatives . . . . . . . . I-60
Correcting Illumination Faults . . . . . . . . . . . . . I-61
Additional Common Problems . . . . . . . . . . . . . I-62
3.11 “Three Paths to Kohler” Summary . . . . . . . . . . . . . I-65
4.8 Basic Microscope Set-up Requirements . . . . . . . . . I-79

I-iii
I-iv
 Preface
*** WARNING – This Book is Different !!! ***
And here’s why . . .
Having spent the better part of my life working with microscopes it has not escaped my attention
that many (if not most) users seem to have difficulties with the same aspects of this discipline,
namely, the true fundamentals. Few of them seem to have any sort of true understanding of what’s
“really” going on, nor of what they ought to be doing in order to achieve better results.
Now, these people are not “dummies,” in fact most of them are quite smart -- so “they” are not the
problem. Further, this basic “knowledge deficit” (for want of a better term) seems somewhat
surprising in view of the larget number of books already published on the subject of Microscopy.
In fact, books on Microscopy appeared nearly as soon as the microscope was invented, going back
as far as the works of the early microscopists, Hooke and Leeuenhoek, for example. So, there are
already lots and lots of Microscopy books out there. Yet, still, most users don’t seem to really
“understand”. . .
Now, all these various books on the subject can be divided into three general categories: the
“Guides” (dealing with microscopy in general, as does this book), the “Cookbooks” (dealing with
“micro-technique” or, the preparation of specimens), and the “Specialty books” (which cover some
narrower aspect of microscopy, but typically in great detail).
Most of the books that the average reader is acquainted with will fall into the first group; the
“Guide” books. And most of these just deal with the same old material in the same old ways. Very
few offer much, if any, genuinely useful, practical advice. So, judging by my personal experience,
it seems that these conventional books are just not getting the necessary “job” done.
And, “Just what ‘job’ is that?”, you may ask . . .
Simple-- the fundamental job of instructing users so that they can get the very best out of the
instrument(s) they have, or are planning to buy. And while the various “guides” seem to appear in
an endless stream, many of those written today are actually classed as “Juvenile literature” making
their suitability for “grownups” a bit questionable. It is in an attempt to address this situation,
based on forty-plus years of in-depth experience, that the present book has been written. So, while
this book is a “Guide,” it is probably not like any that you have encountered before -- it is different!
“And just what’s so different here?”, you ask . . .
Well, as I warned you in the opening above, this book IS different in its approach -- because,
basically, if “the old ways” don’t seem to work, then maybe it’s time to try something new . . .
As you flip through the pages of this book (if you haven’t already) you might have noticed the lack
of the typical (at least, these days) glitzy “stock” photos, for example . . .
This is for several reasons. First, most of these images seem to be just “fill,” merely a blatant effort
to pad out the length of the book; just “clip art” thrown in just to help sell the book and having no
real underlying purpose, other than simply to make the book look more colorful and “attractive.”

I-v
Second, if you check into the photo credits you will see that many of these fancy equipment
illustrations are simply borrowed from manufacturers’ literature, and I don’t expect readers to pay
for something that may soon be outdated, and is usually available for free anyway off the Web.
And, third, you might have noticed that a good many of the photomicrographs (if any) were made
using high-level research scopes (instruments that cost as much as a decent car) and not with
anything the average user is likely to have access to -- so how relevant can such photos really be?
And, finally, just what purpose is a “cutaway” view of the insides of an expensive objective or
condenser (or any of the other usual, similar illustrations) supposed to serve, anyway? Will they
help the reader to achieve a better image? It seems most unlikely -- so, you will not find them here.
Here, “the fill is gone” [sic] and what you should find within the following pages (hopefully) are
many of the very things you really need to know -- and don’t often appear in most other books.
Now, although the underlying facts and fundamental principles remain the same ( “facts are facts
!!!”) their presentation and emphasis may strike you as quite different. In addition, many of the
finer points, rarely stressed in other books, are not only presented here but are also put into a
practical perspective. So, there may be much here that is wholly new to you -- and not found
elsewhere!
Unlike the traditional “textbook” presentations, with this book you will often be “invited” to learn
through actual experience as you progress through the material. (Or, just prove to yourself that the
things said in this book actually are true, and really do work!) And, via this “learning by doing”
process you just might find yourself “well-educated” after finishing this book -- it is intended to
be very much a “hands-on” book, and not “armchair reading” (unless that’s near where your scope
is . . . )
To this end, the all-too-typical “fancy” diagrams have also been purposefully omitted -- after all,
if they didn’t work before, in terms of improving the reader’s understanding, then why rely them
again? So, don’t look for such stuff in this book; things are kept clean, simple and easy to grasp!
Here, you use your own scope, instead of a diagram, and learn by actually using it. In this way, you
will be allowed to discover for yourself just what is and isn’t true (or relevant) and, in so doing
should gain a much better understanding of your equipment and of the practical side of the world
of microscopy. Hopefully, this will lead the users toward refining their skills and enabling them
to enjoy their hobby, and their equipment investment, even more.
Also, it may not have escaped your attention that even the format of this book is somewhat
different -- not simply the content but also “little things” such as the covers, binding, page layouts,
fonts, tables, paragraph styles, etc. Again, all of this is quite purposeful . . .
For example, the covers are laminated extra-heavy stock, hopefully to make them both stain-
resistant and easy-to-clean, because we all know the sorts of things than can happen when one is
busy using a microscope . . . right?
Where possible, information is presented or summarized in table form, for faster reading, to allow
faster comparisons, and to permit quicker back-reference to specific points. Also, critical points
are underlined within the text -- again, for faster “skimming” and easier back-reference (no “Hi-
Liter” needed!).

I-vi
Also, a slightly “larger-than-normal” printing font was chosen to assist the user in reading the
book while using the microscope. The larger font should enable a “greater-than-normal” reading
distance (e.g., from the scope’s eyepiece position to the bench top) so that the reader can more
easily refer to the book while using the microscope!
The overall style of the book is different, too -- more akin to a collection of short essays or a
narrative than to a textbook (and we all know how much fun they are to read . . . ) Long
paragraphs have been avoided so that the reader can more easily “jump around” and then return
to a specific point when reading the book and using the scope simultaneously. (Long paragraphs
might be okay for fiction, or textbooks, but not in something meant to be a fast, enjoyable read,
or easily used as a guide!)
Finally, you may notice that some basic earlier material is reiterated in Chapter 4. This is to allow
some users to “jump ahead” directly to that chapter and apply the methods there immediately,
returning later to read the earlier chapters as “background.” (This recognizes that some people
just prefer to learn that way -- especially when their spare time is limited.)
Lastly, just a wee bit of a caution . . . This book is not “juvenile literature” (not because of the
language . . .) -- it is simply not intended for home-schoolers, nor for those totally new to the
hobby. Much of its content is predicated on the reader already having some basic familiarity with
the microscope (even if it’s only from an old high school or college biology class).
For younger people and older novices there are many other books available which should provide
a valuable, basic introduction to the microscope -- this book would not be appropriate for that
purpose by itself, but should instead be used as a follow-on to such books. (See Appendix II,
Introductory Reading.)
This book is oriented for those who already have at least some experience with a microscope (even
if limited) and yet yearn for something better . . . basically, for “Better Microscopy.”
I hope each of you enjoys reading (and using) this book as much as I enjoyed writing it!

David J. Jackson, 2008.

I-vii
 For a listing of current volumes in
the BETTER MICROSCOPY Series
just visit: www.lulu.com/microscopy

I-viii
1. An Introduction to “Real World” Microscopy
1.1 A “Real World” View of Microscopy
Microscope users vary widely in their levels of understanding of the fundamentals of their
instruments, as well as in many aspects of their use. This book is meant to provide a very practical
(“Real World”) framework for truly understanding the microscope and its use.

NOTE: Throughout this document exposure to and experience with at least one
microscope is assumed. Broad-based knowledge and experience are not necessary;
simply understanding the basic parts and where they are located is adequate.
Knowledge and some experience beyond that, however, may serve to expedite the
learning process. (If some terms are unfamiliar, the reader should refer to
Appendix I, Glossary.)

Users often encounter problems with their instruments, and although these may be nothing more
serious than disappointing results, these problems are more often the result of basic errors in the
setup and/or operation of the instrument than of any actual faults in the instrument itself. They can
usually be traced to either of two basic causes: (a) a lack of “real knowledge,” or, (b) “false
knowledge” (relying on something that simply isn’t so -- “myths,” or misconceptions).
To remedy this situation, we must first examine the factors that govern the performance of
microscope optics, but from a “Real World” perspective. Then we can see how these factors “fit”
into the overall performance of the instrument and how they may affect the quality of our images.
The goal is to enable the user to both achieve better results and to achieve them more easily!
We begin this process by reviewing some of the fundamentals, correcting some common
misconceptions, and then build upon this knowledge to achieve a more complete understanding.
Now, “Microscopy” may be considered the science of studying the “fine details” of objects (by
optical means). To facilitate such studies microscopes were invented and have been developed over
roughly the past 300 years. And while in many respects they may now be considered rather
“common” and familiar to most people, their effective use remains largely limited to a select few.
Still, their power to reveal “hidden worlds” continues to attract fairly large numbers of “non-
professional” users -- those whose professional training lies primarily in other fields, those whose
interest is primarily “artistic” and/or those who are simply fascinated by the things these
instruments can reveal. And while many of these users do manage, perhaps after some struggle up
a steep “learning curve,” to achieve acceptable results, too often many find that the road to real
success is heavily “pot-holed” with disappointments and frustrations.
It is these “typical” problem areas that the following sections are intended to address.
By first dispelling some of the popular myths and misconceptions, and then instilling a greater
level of understanding of the “Real World” principles of microscopy, the path to success should
be made easier and the user’s “success rate” should be greatly improved!

I-1
1.2 Some Popular Misconceptions
First, a big surprise for many of you -- Magnification is simply the biggest, and most serious,
misconception about microscopy! In fact, as we will see shortly, magnification is just one
parameter to consider, and often will be the very least important . . .
While, no one can deny that microscopes are designed to “make things look larger,” this is rarely
their main function. Their “Real World” function is to reveal the fine details of the objects being
examined. It is this ability to reveal fine detail, and not simply raw magnification, that makes these
instruments so very useful, and also so interesting.
Novice buyers of microscopes, sooner or later, seem to yearn for a costly “oil immersion” objective
(usually 100x) in the vain hope that this will magically reveal something that they are “missing.”
But, as is too often the case with uninformed opinion, mostly disappointment awaits and the long-
sought 100x objective, when finally obtained, becomes less and less frequently used.
Properly applied, a good 40x objective can reveal about 90-95% of all that can be seen with a light
microscope! This limit is imposed by the physics of light itself as well as the characteristics of the
objects typically observed. And, for the average user, and the average microscope, the relatively
small advantage of a 100x lens may be even less.
Exactly why this is so will be covered in later sections.

1.3 “Secrets” of Good Microscopy

One of the abilities that separates the skilled microscopist from others who merely “use” the
instrument, is the ability to achieve the best image possible for a given situation. This requires not
only the thoughtful selection of both equipment and methods, but also skilled manipulation of the
instrument itself -- in essence, the practicing of “Good Microscopy.”
No doubt, there are some elements of microscope use that require the accumulation of extensive
knowledge and experience. But there are also many elements which are simply a matter of basic
understanding, and/or of simply exercising the discipline to follow proper procedures -- skills
which most users should be capable of acquiring fairly quickly, and painlessly.
These basic skills fall into two categories:
(1) Knowing how to manipulate the various controls and adjustment of the
microscope in a proper and efficient manner, and,
(2) Avoiding those actions that are likely to either result in deterioration of
image -- or, even damage the instrument.

Unfortunately, those users with limited “Real World” training are at greatest risk for having
developed “bad habits” in their use of the microscope, and so may well require a certain period of
“unlearning” (and practice) as part of their efforts to development new and proper habits. (This is
one more reason why this book was designed especially for use next to the microscope!)

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1.4 Lab vs. Stereo Microscopes
Here we examine the two most common lab microscope types (in their “generic” forms) and point
out some of the more important features, difference, and basic characteristics of each.
To most people, the term “Microscope,” implies one of two forms -- either the “standard”
Medical/ Lab/clinical/research-type (the common “Doctor’s office” type), or one of the true
“Stereo” types.
To reduce any confusion, and provide a clear basis for the current discussion, let’s just label the
first type as the “Med/Lab” (or, just “Lab”) microscope, and the second type as the “Stereo”
microscope.

The major features which differentiate these two basic types may be summarized as follows:

Feature Basic “Med/Lab” Microscope “Stereo” Microscope

Magnification Low to Very High (typ. 4x-1000x)
Objectives Wide range, turret-mounted
(Easily interchanged on turret.)
Eyepieces Typically 10 to 15x.
Resolution Very Good to Excellent
Viewing Monocular or Binocular
(No real “depth perception”)
Image Inverted (“upside down”)
Illumination Simple to Very Complex
(Typ. utilizes a “condenser”)
Very Low to Medium (1x-100x)
Limited choice, often “zoom”.
(May be “fixed” in place if zoom.)
Up to 25x or 30x.
Usually Good, but limited.
True “Stereo” binocular vision.
(Provides “depth perception”)
Normal (non-Inverted)
Usually simple (no condenser)
(Typ. uses simple “top lighting”)

Working Dist. Limited, often only a few mm. Large, often measured in inches.
Objects Prepared Specimens Unprepared (whole) objects.
(Mounted on glass “slides”)

Because of its ability to provide “depth perception,” and the fact that it also has a “normal” (non-
inverted) image, the “Stereo” microscope is used whenever it is desirable to inspect and/or
manipulate a specimen or object under magnification. The long “Working Distance,” provides
ample room between the object and the microscope’s front lens and allows simple “top lighting”
to be used. This microscope is also used when it is necessary to isolate or extract small objects
from larger groups or pieces for the subsequent examination under a “Med/Lab” microscope,
generally at higher magnification.

I-3
The “Med/Lab” microscope, on the other hand, is more adept at the examination of the fine details
of carefully prepared specimens, typically mounted on glass “slides.” Unlike the “Stereo,” the
Med/Lab microscope frequently uses special, often complex, optical methods to reveal the
maximum amount of desired information from a specimen. Its optical system is designed to provide
a high level of “resolution,” and this most often means a short “working distance” -- often 1-2 mm.
(or even less)!
Now, before we leave the subject of the Stereo microscope, it should be mentioned that many of
the former distinctions between these microscopes and the Lab types have become rather blurred,
especially with the development of the modern “mono-objective” research Stereos. Although these
are still mainly characterized by large working distances and “zoom” optics, many of their other
features have become more Med/Lab-like, particularly in terms of illumination methods and their
ability to work with prepared specimens. In fact, for magnifications of 100x (total) or less, there
is frequently a significant overlap in terms of their capabilities (vs. the “Med/Lab” type).
The conclusion to be drawn here is not that one type is inherently “better” than the other, but that
each has a range of applications for which it is best suited. In fact, these two basic types
complement one another very well and are often found side-by-side in the modern laboratory. In
these circumstances the “stereo” type, offers true stereo vision (providing “depth perception”) and
is primarily used for the gross examinations and manipulation of unprepared objects (as this
requires a long working distance), whereas the “Lab” type is most often used for critical
examination of carefully prepared specimens (which requires maximum resolution).

1.5 Lab Microscope Types

Now that we understand the distinguishing features of the “Med/Lab” type microscope (vs. the
“Stereo”) let’s look at some specific types of these microscopes. These can range from the low-
cost “home school” (or, “Hobby”) types (at $100, or less) up to the most expensive research-level
instruments (which can easily cost $10,000-20,000, or more, new!).
These basic Med/Lab microscope types (or categories) may be grouped as follows:
Home School -- Basic, “entry level” scopes intended mostly by use by the school-aged.
Features are limited, and often optics and construction quality are not the best
Classic/Vintage -- These are the older, “professional” or “medical” stands, characterized
by the classic “hinged limb” form of construction. The eyepiece(s) and objectives are
affixed to opposite ends of a body tube that moves up and down for focus. Construction
quality is often excellent and the objectives can be very good.
Modern Lab -- These are of the “contemporary” designs, where the body tube is fixed to
a rigid arm and typically the stage moves up and down for focusing, although on some
models (e.g., AO) the nosepiece may move, either also or instead.
Research -- These are the very “high end” models which offer the maximum range of
features. Construction quality and optics are usually of the highest order. These
instruments often can accept an amazing variety of accessories, although, these are usually
specific to a certain microscope model or series.

I-4
With the “types” now defined, we can proceed to compare them in more detail, as in the
following table . . .

The basic differences between these Med/Lab microscope types may be listed as follows:

Basic Feature Home School Classic/Vintage Modern Lab Research

Coarse Focus Not precise High quality. High quality. Very high quality
Fine Focus Often None Good quality Typ. precision Highest precision.
Condenser Typ. None Simple Abbe Interchangeable Interchangeable
(or single lens) (usually
centerable)
Illuminator None or cheap Uses external. Built-in, often Built-in “ Kohler”
(or attached.) with Field Iris. (real or simplified)

Stage & X/Y Small, no X/Y Good quality. Large typ. has Interchangeable.
(uses “ clips”) built-in X/Y. May be motorized.
Objectives Low-cost Good quality. Higher quality Research quality.
(but not always!) (usually!)

Eyepiece Low cost Good quality High quality Superb quality.

Eye tube(s) Monocular May be binoc. Binoc or trinoc. Usually trinoc.
(or dual-view) (common.) Interchangeable .(1-2 photo ports)
Nosepiece 2 or 3 position 3 or 4 position 4 or 5 position 4 to 7 positions
Interchangeable.
Maximum
Resolution Limited. Good. Depends upon Very highest.
Condenser used.
Photography Difficult. Possible. Optional. Often built-in.
Typical use. Youngsters Amateurs Professional or Research (typ.)
Advanced Amateur
Typical Cost $70-200 N/A $350-3500 $3500 and Up!
(Cost - used): $20-80 $35-500 $200-1500 $600-$2500+
Typ. Methods Brightfield BF, DF, Phase Most. Nearly All.

NOTES:
(1) Except for the “Classic/Vintage” column, the above table refers to new instruments.
(2) The “Home School” column does not include “Classic/Vintage” models.
(3) The “Classic/Vintage” does not include vintage “Research” microscopes (see text).

I-5
Now, we need to examine the basic features of these instruments -- to understand how they
function and thus appreciate the actual differences between them. We can do this best by listing
the most common features and then explaining the “Real World” functions of each. (Refer to the
table on the opposite page.)
As you can readily see from this table, the “Real World” functions listed in the table tend to differ
quite significantly from those that are usually quoted in textbooks. This is simply to create a
smoother transition between conventional thinking and the discussions that follow in later sections.
Now, if you’ll forgive the crude analogy, if the objective is considered as the “heart” of the
microscope, then the illumination may be considered as its “bloodstream.” Unfortunately, very few
books today seem to view things in this perspective -- yet this is very much what it all boils down
to in the “Real World.”

NOTE: It matters very little (except, perhaps, to one’s ego) how fancy the scope is or how
expensive the optics may be, if the objective in use is not set up with proper illumination
you simply cannot get the best results from it. For example, as a perfect “ case-in-point,”
most users would prefer to spend a huge amount of their money on a very high-NA “ Plan
Apo” objective, only to try to use it on a scope with an Abbe condenser -- whereas a
standard objective and a better condenser are actually likely to give much better results!
(All of this will be covered more fully in later sections . . . )

None of this is meant to imply that costly equipment is essential to obtaining good results.
Quite to the contrary, above a certain minimal level of quality, it is entirely possible to obtain truly
excellent results while using rather basic equipment. In fact, the quality of results will usually
depend more on the skills and knowledge of the user than on the size of their budget!
In addition, the more exotic and costly the instrument is, the more difficult it is likely to be to
configure and adjust everything properly. For this very reason, many of the “proud owners” of such
“high-end” machines very often have a smaller, easier-to-use scope handy for use when they
simply want to take a “quick look” at something, and prefer not to be constantly bothered with all
the complexity of the larger instrument! (Didn’t know THAT did you . . . ?)
Which brings up another interesting point -- the “Fun” of microscopy is not directly related to the
cost of the equipment. In fact, beyond a certain point, the higher the cost, the less the fun! For users
with only limited spare time having an instrument that is fun to use can be a major factor in the
overall enjoyment of the hobby. (Ask any professional user who spends much of his time
working with some priceless “dream machine” what sort of scope they actually use at home,
and very often you will find that it’s something simpler and much easier to use!)
Naturally, all of this brings us to the next point -- what should one look for in a microscope?
These days, it seems that the market is absolutely “flooded” with instruments of all kinds, and
these range from the most basic “Home School” types all the way up to some of the very, very best.
How is the average user ever to choose?

I-6
I-7
1.6 Some Things to Avoid
Today, many instruments seem to be built primarily for “minimum cost,” although they are rarely
advertised as such. These instruments are most likely the worst investment. They cost more than
the typical Home School scopes, and, although are less expensive than the “typical” Modern Lab
types, they are also usually of much lower quality and capabilities, due to “Cost Cutting.”
Typical signs of such rampant “Cost Cutting” in these microscopes often appear as follows:
Attachable Illuminator -- These are the really cheap scopes. Instead of making any attempt
to add a proper “ built-in” illuminator, instead, the maker simply throws on a cheap “box light”
to replace the prior (usually) mirror. While this may save some money, it also severely limits
image quality. And they have absolutely no place on a modern binocular microscope!
Stage Condenser -- A one or two-lens condenser that is either “built-in” or attached to the
underside of the stage. It may or may not be “focusable” by twisting in its sleeve. The better
scopes will almost always have a real “substage” which allows proper condenser adjustments
as well as permits the use of alternative condensers when the user’s skills develop.
Attachable X/Y -- This is often a makeshift device added to provide X/Y movement of the
slide. Quality varies considerably, depending upon the maker, but basically it’s now widely
considered to be just a cheap “fix” -- especially if fitted with above-the-stage controls. Better
scopes typically have either a “built-in” X/Y movement, or one of high quality, typically
featuring “Low-position” X/Y movement controls.
Separate Fine Focus -- The “Coaxial” type of Fine Focus, where the Coarse and Fine Focus
controls appear on a common axis, has been around for over fifty years. The use of the crude
“separate” type, where the Fine Focus is located away from the coarse Focus and uses a much
smaller knob is a throwback and a certain sign of rampant cost reduction (on a new scope).
Better scopes usually feature “Coaxial” Coarse and Fine focus controls -- although this is not
always true for the “Classic/Vintage” type scopes.
Screw-type Focus Stop -- This is a “feature” typically created by sticking a 3-cent screw into
the microscope frame somewhere so that a moving part of the microscope can crash into it to
half the upward travel of the stage. Often it has a fancy end on it to make it appear more like
some sort of legitimate adjustment. Over time, these gimmicks can do awful things to the tiny
plastic gears typically found on such scopes. A proper “focus stop” is nearly always built into
the focusing mechanism as part of the original design -- not as an afterthought.

All that said, the encouraging news is that the overall design, construction, and assembly quality
of “ imported” scopes seems to be rising. While “Made in China” scopes were quite problematic
only a few years ago, today many of them represent excellent values, offering good quality and
good optics for a rather low price. However, “good” in this context is not the same as “world-class
quality.” (As used here, “good” really means, “adequate” or, simply “acceptable.”)
This is why the “used” scopes from the major makers (i.e., Olympus and Nikon -- if marked,
“Made in Japan”) still usually sell for much more than the nearly-identical-looking newer “copies”
from the “unknown” makers -- that’s the difference (in quality) between “okay” and “very good.”

I-8
Other advantages of “used” Modern Lab scopes (from major makers) are the availability of parts
and service (albeit, costly) and the general availability of accessories for these models. Also, these
models tend to retain more of their value than the lesser-known “stuff.” So if you should, at some
point, decide to resell your Nikon/Olympus/Zeiss/Leitz (etc.) you may well get back close to what
you paid for it. However, reselling some “unknown” is a much more speculative undertaking.
So, aside from problems due to excessive “cost cutting,” many (but not all) of the otherwise
acceptable scopes from these newer makers also seem to exhibit similar weaknesses. Some of these
are correctable with minimal difficulty, but others are not.
The consistent weaknesses of these newer “Unknown Maker” Lab scopes seem to be:

Cheap eyepieces --Image quality may sometimes be improved simply by switching to

moderate-cost, higher-quality eyepieces (the objectives are usually less of a problem).

Cheap Condenser -- The excessive use of plastic seems to be common in many of these
scopes, particularly in part which holds the condenser iris. Plastic here is a warning! Also,
many of these cheap condensers are non-divisible. Since the top lens often cannot be removed,
applications requiring much low-power work (e.g: 4x objective) may be compromised.

Poor Illumination -- Problems here include serious “hot spots” (uneven illumination), and a
small light port diameter (limiting the visual field at low power). Before you “upgrade” one
of these scopes with wide field eyepieces, be sure the illuminator (and the condenser) can
cover the larger visual field at low power (4x). Many cannot -- so check first!

Wrong Condenser Height -- Even worse than above, it appears that on many of these scopes
the condenser is simply too short to be focused properly -- it may be impossible for the user
to get the condenser close enough to the slide for it to work properly with even a 40x
objective.*

Poorly Designed Condenser Mounts -- In addition to often requiring a “nonstandard”

condenser (typ. 37mm O.D., and 27-33mm “ tip-to-flange”) it seems that many of these mounts
also lack a proper “ focusing stop” and, instead, appear to rely on simply allowing the
Condenser mount to bump (smash?) into the underside of the stage!

Binocular body quality -- Although outwardly decent-looking, the internal optics may be of
questionable quality. One sure sign of cheap optics is different colorations between the two
eye tubes, although this may range from barely detectable to the very obvious!*

*These are serious problems -- adequate grounds for rejecting a scope that has them!
(However, not all “ unknown maker” scopes have problems -- a few seem problem-free.)

In general, the problem of condensers and condenser mountings will become more significant as
your knowledge and skill level increase. Few new users worry about such things simply because
they do not recognize their importance to achieving really good “brightfield”images.

This “Condenser Height” problem is clearly a serious “defect” in the design of the scope. For
maximum NA, the Abbe condenser must be nearly touching the underside of the slide. Yet, on

I-9
some of these scopes, there is such a big gap there that even immersion oil will not stay put. Often,
there is no simple way to fix this problem -- the condenser provided with the scope is simply
not right.
One consequence of this fault can be that, initially, there may appear to be good contrast with the
objectives up to 40x, but that the 100x never seems to give a good image. This is because a
Condenser that is “too low” cannot deliver anywhere near its full NA. In addition, because the
condenser focus is incorrect, the condenser iris cannot properly control image contrast and “glare.”
The importance of the condenser is more apparent with “Research level” scopes, which offer a
wide range of condensers to choose from, for various applications. The excellent results obtained
with these scopes are only partly due to their expensive optics – it is also due to their high-quality
condensers, which can deliver the best possible illumination!
Thus, one of the most significant differences between the “Research” microscopes and the ordinary
“Med/Lab” microscopes is their condensers. This is why putting high-NA objectives on an
ordinary scope often fails to achieve the desired results -- you simply can’t get the illumination
“quality” needed when using just an ordinary “Abbe” Condenser!. (All this is covered more fully
in Chapter 3.)

1.7 Research and Vintage Microscopes

So, let’s continue with examining the modern “Research” microscope. Unfortunately, this term,
“Research” has become greatly overused (particularly on some popular auction sites where it
seems to be applied to just about everything). In its proper sense, it denotes a microscope with the
capability to function at (or very close to) the “state-of-the-art” level.
Still, there are some distinctions which may be made in order to usefully distinguish these. If we
assign categories such as Basic, Intermediate, and Advanced to the general “Research-scope”
classification, we can then summarize the characteristics of each version (or, “type”) as follows:

Function/Feature Basic type Intermediate type Advanced type

Fine Focus 200u per turn 100u per turn 50u per turn
Illumination No Field Iris Simplified Kohler True Kohler
Condensers Basic types Advanced types Advanced types
Nosepiece(s) Typ. fixed. Interchangeable Interchangeable
(4-5 position) (4-6 position) (up to 7 positions)
Accessories Few, if any. Reasonable Very Extensive.
Typ. Weight Moderate Fairly heavy Absolutely Massive*

* (“If you can move it without a truck, then it probably isn’t a real Research scope!”)

I-10
As can be seen from the above, the overlap between the “Advanced” Research types and the lesser
scopes can be considerable, except for the “Fine Focus.” This tends to be the real demarc. You can
quibble about other points, but the accuracy of the Fine Focus is usually marked right on the knob!
Only an instrument intended primarily for research is likely to have a 50u/turn (or 100u/turn) fine
focus. These high-accuracy focusing controls are only needed for precise techniques, such as high-
resolution photography with the very best optics. For general lab use, however, they are usually
just too “slow.”
Now, having covered the “Modern” scopes (Med/Lab and Research) in some detail, let’s turn our
attention to the “Classic/ Vintage” category -- the scopes that everyone “knows.”
The “Classic/Vintage” microscope category may also be split into “subgroups,” which can be
summarized as follows:
Antique -- As “collector’s items” they are not covered here, and are not all that useful.
School types -- These include the very basic “Elementary School” types (no condenser),
the “High School” types (no oil immersion lens), and the “College” types (with oil
immersion). Occasionally these have inexpensive, low-power illuminators in place of the
usual mirror.
Clinical/Medical -- These are the classic “Doctor’s office” scopes and may be identical
to the “College” (or, “Medical School”) type listed above. The black-and-chrome scopes
of this type made by the American makers (Bausch & Lomb and AO/Spencer) in the late
1940s and 50s were excellent, and typically of high mechanical and optical quality.
Research -- These are rare, but highly desired by both collectors and knowledgeable users.
In their day they were superb instruments, designed to rival the best microscopes from
Europe and they often sold for more than the cost of a new car (back then, anyway). Many
still have their original “Apo” ( “Apochromatic”) objectives and high-quality condensers.

NOTE: Most of these scopes were intended for use with an external “research
illuminator” (or, “lamp”) of some type, and when so configured they could produce
amazing image quality, although usually required some skill to achieve. (One of a vintage
Research scope is the excellent Leitz Ortholux-I -- a fine older scope, but one also
featuring a “built-in” illumination system and a more modern design.)

Of these, the School types are the least useful, except perhaps for “Home School” uses where their
robust construction is a benefit -- just don’t let your kid drop one of these on their foot! (Unlike
some of the modern stuff, one of these older stands could do some serious damage . . . )
Now, the Clinical/Medical stands are a bit more interesting. Here we have “Real” instruments
which are available in nearly endless supply (tens of thousands were produced) and which are also
frequently available at low cost. The reasons for this are that they typically lack any form of built-
in illumination, and are thus “out-of-fashion.” Also, most users have absolutely no idea how to set
up one of these properly, and thus can obtain only disappointing results. However, this is rarely
the fault of the instrument (unless it is in poor repair and/or the optics are damaged).

I-11
The novice user should avoid the oldest versions of these, usually having “brass” lenses, and
perhaps even brass bits showing. A better choice are the “black-and-chrome” models as these
usually date from the 1950s (or later) and frequently have improved optics. One of these, in really
good condition and made by a “top name” maker, can often be found for about half the cost of a
Modern equivalent made by one of the “ unknowns” -- and the older stand will probably still have
lots of useful years remaining, something that can’t be said with any confidence about the newer
stuff.
This leaves only the “vintage Research” scopes -- but, as some might argue, some of these may be
among the very best scopes of all!
Here, again, the “flood” of new scopes from overseas has resulted in depressed prices for all but
the very best and most desirable vintage Research scopes. Many of these have all the advantages
of their less-costly Clinical/Medical brethren, but also have many of the types of features that make
Research scopes in general so desirable. One excellent case-in-point is the Leitz “Ortholux-I”.
While considered a “ Vintage” scope because of its age, the “Ortholux” nevertheless offers many
of the features of its newer cousins -- built-in illumination, interchangeable nose-pieces,
interchangeable stages, interchangeable eyepiece tubes and a wide range of condensers, etc. And
these can usually be obtained for little more than the cost of a good, used “Modern Lab” scope.
One additional advantage of scopes like these is that they can be “built-up” over time, by adding
accessories as the need arises (or the money becomes available -- we all know how that is!)
Thus, you can start with a basic instrument and slowly add new optics and accessories so as to
continually enhance the capabilities of the instrument as your needs and skills grow. This option
is typically not available with lesser scopes. But, if you should elect this route, just be sure that you
choose an instrument that was fairly popular in its day, so there is a high likelihood that the parts
you will want can still be found relatively easily.
At this point we have progressed from the lowliest scopes (often having no fine focus at all) to
some of the very finest instruments made, hopefully, imparting some understanding of the
differences between them and some of the significance of these differences. Later sections will
examine some of the points raised here, and some not mentioned, in greater detail.
However, the following section attempts to summarize the material just presented and to frame it
in a context more immediately applicable to the individual user.

I-12
1.8 What Defines a “Good” Microscope?
Not surprisingly, just as the microscopists (users) themselves vary in terms of their skills and
abilities, the instruments also vary in terms of their capabilities. And in this connection another
myth arises, that the instrument itself is all important and the skills of the user mean very little.
To some extent this “machine” fallacy is widely fostered by the advertising claims of those
manufacturers who would have one believe that simply by purchasing their latest “wonder-scope”
all of your microscopy application problems will be solved -- simply and forever!
So then, you might ask, “Just what IS a good microscope . . ?” Ahhh, glad you asked . . .
This brings us to the point of this section, namely, that a “good” microscope is any one which
satisfies the minimum requirements for the application at hand. Thus, somewhat surprisingly, it
is possible for even an older, well-used “vintage” instrument to be just as viable as the very latest,
ultramodern, automated wonder-scope -- for some applications (but, not for everything) !
Determining just what constitutes the “minimum requirements” is one of the skills exhibited by
a good microscopist, but is also a skill that can be acquired by any user with a good basic
understanding of the “Real World” fundamentals.
The following table summarizes some of the more important features which are likely to effect the
basic selection of an instrument:

Desirable Acceptable Undesirable

Coaxial Focus Controls
Condenser NA>0.9(dry)
or NA>1.2 (oil type).
Focusing Condenser
(Rack-and-Pinion type)
“Kohler” Illuminator*
(Field Lens and Iris)
Halogen Lamp (6-12V)
Built-In X/Y Movement
(Low-position controls.)
4 or 5 Position Nosepiece
“Made in Japan” or
Germany, etc.
Separate Focus Controls No Fine Focus at all.
Condenser NA>0.65 Condenser unmarked.
(Simple, “dry” type.) (or No Condenser at all)
Focusable Condenser Fixed Condenser
(Spiral-sleeve type) (or No Condenser at all)
“Critical” Illuminator Simple Illuminator
(or, Field Lens, No Iris) (No Lens and No Iris)
Standard Lamp (120V) LED or Fluorescent.
Attached X/Y Movement No Movement (manual)
(with Stage-level controls.)
3-Position Nosepiece 2-Position Nosepiece
“Made in China” (new) “Made in China” (older)
(some are now quite good). or an “unknown” source.

* Many makers claim “Kohler” Illumination, but this is actually rarely true.
Usually what is provided is a “simplified” form of Kohler illumination, very
close to “Critical” illumination. All this is covered a bit later, in Chapter 3.

I-13
1.9 Pursuit of the Perfect Image
The preceding discussions have naturally led us to the somewhat controversial subject of
microscope “upgrading,” or, as some may prefer, the “endless pursuit of the perfect image.”
Now, what many users are actually seeking when they begin this process is often merely relief
from the disenchantment they are experiencing with their current instrument (and its optics). More
often than not, the scope’s optics are blamed for every perceived image problem . . .
This “need to upgrade” is usually based on loosely perceived notions of inherent “faults” or
limitations, hopefully to be remedied by just making some “improvements” in the equipment --
either additions or replacements. However, unless executed thoughtfully, such “upgrading” is not
very likely to result in anything beyond just the transient joy of having acquired something “new.”
Many users, for example, almost as soon as they receive a new instrument, seem to begin to
imagine the benefits of “upgrading” to “better” objectives. This usually translates into either higher
magnification or higher-NA, or both. (Novices usually opt for more magnification while the more
experienced users often wish for higher NA’s, and thus hope for the ability to see finer details.)

Note: “NA” is simply a measure of the ability to see fine detail. (See Secs. 2.3 and 2.5.)

Now, this “more Magnification” route is self-limiting -- a 100x objective is about tops for a
standard microscope. And, for those who may think that switching from a 95x objective to a 100x
will make a difference, this has almost exactly the same effect as simply pulling the eyepieces out
about 3/8-inch. (Try it -- and see how little difference it really makes!)
Further, as discussed later in this book, the improvement in performance of a 100x “oil” objective
over a 40x “dry” objective is typically much less than one might normally expect. While the image
is larger (or, “more magnified”) the actual “resolution” is not all that much better.
“Resolution” is one reliable measure of performance, and represents the ability to see fine detail.
This is largely determined by the “Effective NA” of the objective, which, in turn can very easily
be reduced from its marked value by improper use. In fact, higher-grade (high-NA) objectives can
only achieve their maximum performance when operated under carefully controlled conditions,
and especially the “conditions of illumination” (and this includes the condenser adjustment).
Unfortunately, this is precisely one of the major weaknesses of the common “Abbe” type
condenser, found on about 98% of all Lab microscopes. The performance of the “Abbe” begins
to decrease when it is used at NA’s higher than about 0.7, or so. This means that as the objective
NA rises above that level, the Abbe condenser becomes less and less able to make the objective
work at it’s maximum NA. Thus, the concept of “Effective NA” -- or, the “usable” resolution
of the objective under its actual conditions of use.

The inability to effectively support high NA objectives (e.g: NA>0.65 or 0.70) is why it is
generally inadvisable to try to use very high-NA objectives on scopes equipped with just a basic
Abbe condenser.

I-14
The Resolution Limits for some common microscope objectives are as follows:

Objective Maximum NA Resolution Limit* “Abbe” Limit

4x 0.10 0.00325 mm 0.00325 mm
10x 0.25 0.00130 mm 0.00130 mm
20x 0.40 0.00080 mm 0.00080 mm
40x 0.65 0.00050 mm 0.00050 mm
40x 0.95 0.00035 mm 0.00040 mm
100x 1.25 0.00026 mm 0.00030 mm
100x 1.35 0.00024 mm 0.00028 mm

* “Theoretical” limit. However, worse than this above NA 0.65 with an “Abbe” condenser,
as listed in the last column. The reasons for this are covered in Chapter 2, and Section 3.5.
As may be seen in the above table, there is actually not much to be gained (in terms of the actual
resolution) by attempting to utilize an expensive high-NA objective on an ordinary Lab scope.
While there is always some improvement with increasing NA, this is usually less than one might
hope for -- due to the limitations of the Abbe condenser, and sometimes even of the scope itself.
Fortunately, most users have no real need for high-NA optics (alluring though they might be) and
most Lab microscopes are actually incapable of fully utilizing many such lenses (as was mentioned
above) even if they were installed -- and this is especially true for those low-cost scopes equipped
with just simple, Abbe condensers and basic illuminators (typically, lacking a “Field Iris”).
In fact, these factors (above) can be such a significant limitation that many might be simply better
off learning how to get the most out of their scope, using just “ordinary” objectives, gaining skills
and experience in the process, before actively considering more “exotic” optics!
So, before the user embarks on what may very well prove to be a costly and fruitless effort, it
makes sense to review some common limitations of most Med/Lab microscopes, and their
remedies:

Limitation Effect Solution or Remedy

Objective(s) “Color fringes”. Change to matching eyepiece(s).
Poor “resolution” & Verify illumination, then check for
good image contrast. condenser focus error (too low).
Poor “ resolution” & Verify illumination, then check for
poor image contrast. “coverglass thickness” error.
Eyepiece(s) Low-contrast image. Check illumination for “glare.”
Dark spots in image. Check eyepieces for dirt, etc.
Lack of Field iris “Glare” in image Reduce NA (with condenser iris).

Now, you might have noticed that this is just a “short list.” And that is intentional.
There really are very few genuine problems that can be solved simply by “upgrading.”

I-15
Most users’ problems are actually due to lack of skill and/or set up errors in the use of the
instrument. The number of “legitimate” reasons for attempting an “ upgrade” are few, and include
mainly switching to “Plan” optics, adding a camera port, or changing to a higher-quality condenser.
Also, note that “upgrading” is a very different matter from adding new capabilities to the
instrument -- such as darkfield and/or phase contrast imaging. In such cases the user is now adding
accessories to extend the capabilities of the instrument, and this is not at all the same as blindly
swapping components in an effort to remedy some perceived fault, or “improve” the image. (Of
course, any components that actually are defective should be repaired or replaced -- first!)
So, the problem now becomes one of differentiating between any real limitations imposed by the
equipment and those simply imposed by the current limits of user’s skills.
After all, if the user cannot fully exploit the equipment he or she already has, what is there to be
gained by “upgrading” to equipment that is likely to be even more demanding?
On the other hand, if the equipment actually is at fault, how is the user ever to gain improved
skills? And, worse, how is the average user ever to know the difference?
Fortunately, that’s easy -- simply “verify” the proper functioning of the instrument!
This is a simple process, and, if only to put users’ fears at rest, checks to “verify” that the existing
components are functioning properly. And, it also can often identify those that are suspect.
However, this is only a reasonable thing to attempt if there is some assurance that not only is the
instrument otherwise in good working order, but the user is skilled enough to be able to “push” the
objectives toward their limits for testing. Needless to say, this is a bit of a “Catch-22" situation!
This last concern can be addressed simply by adopting a methodical approach.
Better yet, this process of “verifying” that everything is in good order can easily be extended to
one of “optimizing” the scope to ensure that everything is in the best possible operating state!
The following section will provide the user with a number of simple methods with which to
evaluate the capabilities of his or her current instrument, and perhaps allow them to refine their
skills just a bit in the process. In some cases, this process may even make “upgrading” entirely
unnecessary . . .

I-16
1.10 “Optimizing” Your Scope
Optimizing is simply the process of confirming that the instrument is properly configured so that
it can consistently deliver its maximum useful potential. Much unlike so-called, “upgrading,” the
process of “optimizing” is basically just “checking” things, and can often be accomplished for free!
In optimizing, we are concerned with the three most critical components of the microscope imaging
system, namely: the objective, the illumination (illuminator and the condenser), and the eyepiece,
usually in that order of importance. However, we must start with the eyepiece since we will need
to use that to check everything else.
Naturally, with eyepieces being so exposed, they often collect a lot of “stuff” on the eyelens. And,
to proceed, these must at least be clean. So, the first thing to do is check for cleanliness.
You can do this easily by simply rotating the eyepiece slowly as you look into the instrument. If
anything in the visual field “ follows” the rotation, then that is in the eyepiece and usually on the
eyelens. (However, a few tiny spots won’t bother anything -- it doesn’t have to be perfect, just not
obviously dirty or greasy. There’s no point in going nuts here, “clean” really doesn’t last long).
If you do need to clean the eyelens use a solvent and use only a clean, soft cotton cloth or soft,
cotton-tipped applicator. The safest solvents are 95% Isopropyl alcohol or Naphtha (“lighter
fluid”), but even these should only be used sparingly. (Use CAUTION , these liquids are
flammable!)

NOTE: Never use any “window cleaner” or cleaners for eyeglasses. These can attack the
optical coatings and/or the optical cements used in eyepieces, causing permanent damage.
Always wipe optics gently when cleaning -- never apply pressure against a lens!

Now, concerns about eyepiece performance can be addressed by two simple tests.
Assuming that it is at least “clean enough,” the next step will be to test this eyepiece simply by
using it as a “hand magnifier.” Hold the eyepiece “inverted,” with the eyelens facing the object and
the open end toward the eye. The critical area for this test is the center region, about 1/3 (or a bit
less) of the total field. Here we are looking for a sharp image with good contrast, with object edges
well defined. You may ignore any curvature or colorations that occur outside of the central area --
at this stage we are simply checking for a good, sharp center image.
Once you are satisfied that the eyepiece can function well as a magnifier (if your scope has two,
they should both be checked), you should replace the eyepiece(s) in the instrument and proceed
to the next set of tests.
But, before we can actually test the remaining microscope optics (condenser and objectives), we
need to verify that the illuminator is functioning acceptably. This means that it has a uniform
output and is capable of covering the full visual field at low power. (If there is a Field iris, it should
be opened fully. If there are other attachments, such as an Auxiliary Lens, this should be checked
to verify that it is properly in place.) Note that the following test is only for “built-in” illuminators.

I-17
To check the illuminator we first turn it on (duh . . .). We next place a piece of white paper or a
thin white card over the top of light port so as to reveal the illumination pattern. (This test
“target” should be large enough to fully cover the light port opening.) The illumination
intensity (brightness) is then adjusted so that the pattern of light from the illuminator may be easily
seen.
At this point there should be a circle of light visible on the paper target and this should be quite
uniform over most of its area, although some darkening at the very edges is acceptable. (If the
illuminator has a “lamp focusing” adjustment, this may now be manipulated to verify the setting
at which the illumination appears most uniform..)
However, if the illumination pattern shows a “hot spot” in the central area, and this cannot be
adjusted away by lamp focusing (or there is no focus adjustment), then there may be a problem.
One common problem (mostly with used scopes) is that an incorrect lamp has been installed.
Microscope lamps often have special filament designs specifically designed to provide uniform
illumination, but this adds to their cost. So, lamp “substitutions” are often encountered.-- usually
just to save money. Also, with older scopes, the correct lamp may no longer be available.
For either of these reasons, cheap substitute lamps often appear in such instruments, and these
quite often yield unsatisfactory results. In any case, if the illumination is not uniform you should
verify that scope does have the correct lamp installed. (If the instrument does not have this
information marked on it, you may need to contact the distributor or one of the many microscope
lamp suppliers on the Web, many of whom have lamp reference catalogs available online.)
Now, with the illuminator operating acceptably, remove the paper test target, check that the
eyepieces are properly installed, and move the 4x (lowest power) objective into position. Next, this
objective is focused on any convenient specimen, and once focused, the specimen slide is removed
from the scope’s stage, leaving an unobstructed view of the top of the condenser.
The condenser iris should be opened fully, and the condenser raised to its highest position, but not
above the level of the surface of the stage. (The condenser should “stop” just below this point, if
it does not, then there may be a “stop” problem -- which we will address a bit later.)
At this point you should be able to verify that the field-of-view for the 4x objective is fully and
uniformly illuminated. (On inexpensive scopes, the “uniformity” may leave a bit to be desired.)
This test assumes the use of a “standard” NA1.2 to 1.30 Abbe type condenser. Other types may
give slightly different results. “Swing-out” condensers, for example, should be checked with their
top element moved out of the light path. Also, on scopes equipped with high-NA condensers (NA
1.35 or more), these may not be intended to provide coverage of such a large field, since many of
them are intended specifically for use with high-NA (and high magnification) objectives.
If the illumination is reasonably uniform, but the illumination does not cover the entire visible
field, this may or may not be a problem.. First, if the “circle-of-illumination” is concentric with the
visual field limits, then the problem may be just that the “field coverage” provided by the
condenser is inadequate. (If the condenser is “divisible,” you should remove the top and re-check.)
But, if this “circle” is “off-center,” there may be cause for concern. If so, then first check to verify
that the condenser is fully and correctly seated in its mount. If so, next check to see whether the
problem is the condenser -- verify that the condenser iris actually is fully open. (If not, open it!)

I-18
If neither of these actions corrects the problem, then there may be a significant fault with the
instrument itself. To test this, begin by making a small “X” mark in the center of the paper test
target be began with. The position the target on the illuminator light port so that the “X” is in the
center of the lighted area. Next, using the condenser only, lower the condenser until the “X” is seen
to be in focus in the scope’s eyepiece(s).
Now, determine whether the “X”, positioned in the center of the lighted area, is also in the center
of the eyepiece field, or not. If not, then the scope’s arm (and or/substage) may simply be out
alignment with the illuminator light port. If it is, then the problem is most likely one of “coverage”.

NOTE: However, note that some scopes (Zeiss, for example) rely in the use of an
Auxiliary Lens in order to provide full field coverage at low power. If you have a scope
of this type, and this lens is missing, then this can also cause this test result. Unfortunately,
sometime these lenses were part of the scope, held in a swinging holder beneath the
condenser, and sometimes they were mounted into the base of the condenser itself. Other
scopes, such some of the older Nikon’s and the Olympus BH series used separate
Auxiliary lenses. In any event, if the lens is missing you cannot get full use out of the
scope!

If everything seems correct – both the Field Iris (if any) and the Condenser Iris fully opened, the
condenser is fully and correctly seated in its mounting and the condenser is set to within a “slide
thickness” (1mm), or less, of the stage surface, and there is still inadequate illumination coverage
of the visual field, the problem may be the eyepieces. Check to see whether the scope is equipped
with “Wide Field” type eyepieces. If so, and these were not originally provided with the
instrument, then the problem may simply be that the scope was never designed to work with these
eyepieces. This is especially likely if the illumination extends very nearly (but “not quite”) to the
edge of the field and the scope is not of very recent design.
Once this test has been completed (whether successfully or not), the condenser should be set to its
upper position (just below the stage surface, as before), and the paper test target removed. We are
now ready to begin testing the objectives.
For the objectives we use a simple test, which might be termed, the “centerfield sharpness” test.
Regardless of the objective “type” (simple achromat, plan achromat, or whatever . . .) the image
produced in the central area of the visual field should appear quite “sharp,” and should also exhibit
a reasonable contrast level. If an objective fails to do even this, then something is really wrong!
To test the scope’s objectives we will also use only the “central field” of the eyepiece, which we
have already checked (above).
We begin by using the “10x” objective, since that is relatively insensitive to the illumination used
(and we would like to have only one “variable” at a time to contend with). With this objective in
position, and an object in place on the stage, set up the illumination as you normally would. Be
sure the condenser is raised just beneath the slide (without quite touching it.)
If you wish, you may skip ahead to Chapter 4, briefly,to review setup and illumination.

I-19
Focus on the object (any well-stained object slide, or, even a bit of newspaper or magazine photo)
and adjust the condenser iris to the point where the image just begins to darken a bit. This should
result in a sharp image with good contrast. (If not, the 10x objective may be dirty, or even defective
and should be removed and examined). However, if the image looks acceptable at this point then
it’s time to move to the second half of this test, which might require a bit of practice.
Observing through the instrument, displace the objective slightly by rotating the nosepiece just a
tiny bit away from its detent. The goal is to shift the details which were in the center of the image
to one edge of the field, and thereby shift the details from the opposite edge into what is now the
new center of the field. Minor refocusing maybe required here to get the sharpest image. (Note:
this is NOT the same as shifting the object to achieve the same image -- that would accomplish
nothing. For this test we must shift the objective, relative to both the eyepiece and the object.)
The purpose of this maneuver is to use the “central area” (or, “optical axis”) of the eyepiece to
view the edge of the image from the objective. With a decent objective, the image quality will be
very nearly the same as before. If not, then this raises questions about the quality and/or condition
of the objective. This test may be repeated with the higher-power objectives, although those may
well require a little patience to get the image shifted as required, and other factors may enter in.
Also note that this test is made somewhat more severe if “Widefield” eyepieces (FN = 20) are
used, since these will entail shifting the objective even more (due to their extra-wide fields).
For eyepieces and objectives of good quality it will be found that the image sharpness (and detail)
that can be obtained close to the edge of the field (perhaps 3/4 or more of the way from the center)
may be very nearly as sharp and detailed as that obtained near the center of the field, provided only
that the image is simply refocused. (However, even here the exact edge is a different matter,
especially if any color band exists -- unavoidable in some designs, even very good ones.) The sole
advantage of “ Plan” optics is that they include optical compensation which removes this need for
refocusing, and thus nearly the entire field will be in focus simultaneously (for flat objects).
This testing may be repeater with the high-power objectives, but, before that may be done, it is
necessary to return to the condenser and verify that it is functioning correctly.
Common problems with the standard condenser are improper condenser height adjustment
(“focusing”) and/or improper setting of the condenser iris. (It is also possible that the condenser
height may be incorrect for the scope -- See sec. 1.5, page 9.)
Still, the most common “ condenser problem” is simply that many users just do not (or, for one
reason or another, cannot) focus it properly. (Hardly their fault since few books or manuals really
instruct them on how to perform this important task effectively. -- except. perhaps, this one!) And,
if the condenser is not focused properly, it can be nearly impossible to obtain a good image with
the 40x and higher objectives. So, we need to check the condenser . . .
For this “Condenser Test” all that is needed is a “frosted end” microscope slide. (It doesn’t matter
whether there’s any specimen on it since all you need is the frosted end. If you don’t have one
handy, you can mount a thin piece of white paper, about ½-inch square, onto a plain slide using
transparent tape and simply use that instead. Just be sure that the paper lies perfectly flat on the
surface of the slide.) Now, armed with your new “Test Slide,” here’s how to conduct this test . .
.

I-20
Place the slide on the stage with the frosted area (or paper) facing up, and centered over the
condenser. With the illuminator on, raise the condenser so it’s just barely below the slide. With
the condenser in this position you should see a sharply defined spot of light on the frosted area. (If
the scope has a Field Iris, this should now be opened fully -- for now, we won’t worry about that
Iris.)
Now, if the spot is not sharply defined, or you are unsure, simply lower the condenser slowly.
If the spot gets sharper, the condenser was too high. But, if the spot becomes even less sharp, then
the condenser was not high enough. (The normal height is about 1/2 “slide thickness,” or less,
below the bottom of the slide, for a “standard” NA 1.2 to 1.25 Abbe condenser.)
However, if you find that the microscope will not allow you to raise the condenser to the required
position, then either the condenser stop is set too low or the condenser is for some reason too
“short” for your microscope. This may be because the condenser is not properly positioned in the
condenser mount, or perhaps because of an Auxiliary Lens problem (incorrect or missing).*

*NOTE: Some scopes have condensers that are designed to work in conjunction with an
Auxiliary Lens. (Examples are the Zeiss Standard/Universal,Photomic series and Olympus
BH series.) If you have such a scope and the Auxiliary Lens is missing, (or the condenser
or Aux lens is wrong) then the condenser simply will not focus properly until this problem
is corrected. But, if your scope passes all these tests, then there’s likely nothing to worry
about.

With the condenser at this starting point, open and close the condenser iris while observing the
light-spot. It should lighten and darken almost uniformly, without changing size, as the condenser
iris is opened and closed. (This confirms that the condenser height is roughly correct.)
If the size of the spot changes as the Condenser Iiris is operated, then the condenser height is off.
If the test slide is oriented correctly (frosted side up) and the spot size is changing, then this
indicates that the condenser is too low (it should be quite near the underside of the slide, as above).
The “normal” position for the standard Abbe condenser (“dry”) is not more than 1/2 “slide
thickness” below the underside of the specimen slide. This is an acceptable position for most
routine work and a good starting point for more exacting adjustments. (More precise methods for
condenser adjustments may be found in Chapter 3.)
Now, if your scope has passed all the above basic tests, then it is quite likely to be in very good
condition (at least, optically) and capable of providing good image quality in most cases.
If you wish, you may repeat the 10x objective test (above) using higher power objectives, although
the “displacement” test may be difficult with these and is not absolutely necessary. (For a review
of setup requirements for the 40x and 100x objectives, see Chapter 4, Sections 4.8 and 4.9.)
More factors that can affect microscope image quality, and their effects, are discussed in the
following Chapter . . .

I-21
Notes

I-22
2. “Real World” Microscope Optics
2.1 “Real World” Optics -- a Demonstration
Image-forming optics are composed of one or more lenses. Here, we will begin with just a single
simple lens. This basic optical device will allow us to develop a “feel” for what real lenses
accomplish, how they function, and what limitations they typically possess -- all considerations
important to the “Real World” understanding of the microscope.
For this demonstration we will use a semi-darkened room (excessive room illumination will only
make things we are trying to see a bit less apparent). For our “test object” we may use either an
outdoor scene (through a window), or an indoor scene (as with a lamp on a desk, or a floor lamp).
The only other items required are a simple lens (a hand magnifier or “reading glass”) and a sheet
of white paper or similar material to use as a “ screen.”
Now, everyone “knows” what’s about to happen, or do they . . . ?
As one might well expect, the lens may be used to form an image of the selected test object on the
“screen.” In this case, the screen is positioned with its flat side facing the test object and the lens
is interposed between the test object and the screen, more or less parallel to the screen. As the
distance between the lens and the screens varied an “ image” of the test object will appear on the
screen, its sharpness determined by the distance between the lens and the screen.
So far, all “textbook” stuff and very boring, right . . . ? Well, maybe not . . .
With the lens held so as to form the sharpest possible image on the screen we may begin the “real”
learning process. Study the image and note that (a) the image is inverted, and (b) the region of
maximum sharpness seems to be limited to the center of the image. Also notice that if the screen
is moved close to the lens there is a “circle of illumination” near the lens which gradually
diminishes until it becomes the image some distance away (lens held steady and only the screen
moved).
We have now observed the basic operation of a simple lens, bringing the light rays from a distant
object to a focus, forming an inverted image of the test object on a screen. No big surprises here --
however, now we begin to enter the “real world” . . .
Up to this point the center of the image, the center of the lens, and the test object have all largely
been aligned on along the “optical axis” of our simple system. This is how textbooks like to show
things, in the simplest possible way. Unfortunately, the “ Real World” is just not that simple. . .
Now, to show some “Real World” effects we need to begin moving things “off-axis.”
Holding the relative positions of the lens and screen constant, slowly shift them so that the image
of the test object center is shifted toward one edge of the screen and note that it is no longer
possible to obtain a sharp image of that part of the object simply by moving the lens back-and-
forth. Then, try rotating the screen slightly so that it is no longer parallel to the lens. With a bit of
experimentation you should observe that the “off-axis” image can be improved somewhat by
altering the lens-to-screen distance and also by changing the orientation of the screen.

I-23
If the test object contains a light source, such as a bare light bulb, you may also observe some
rather strange shapes occur as the “off-axis” focusing process is attempted. The net result of all this
is that you have just observed the major defects of all simple lenses: coma, astigmatism, and
spherical aberration. All lenses exhibit these optical defects to some extent, and it is a big task to
reduce them to an acceptable minimum when designing a lens system for a given purpose.
One other important characteristic of a lens (any image-forming optical system) lies in its
“coverage.” In the above experiment you may have observed that the “area of sharpest definition”
for the image (or, its image coverage) was somewhat less than the actual diameter of the lens used.
Also, you might have observed that areas of the test object which were not exactly in the image
center, and therefore were a bit out-of-focus, could perhaps be brought into focus by slightly
altering the “lens-to-screen” distance. Thus, different parts of the test object reach focus at slightly
different distances from the lens.
What this shows is that, for a simple lens at least, the “image plane” is not really a plane at all but
is actually a “curved” (3-dimensional) surface. This is one more problem that optical designers
must contend with, and one more parameter that distinguishes the quality of an optical system,
namely the ability to form a “flat” (“Plan”) image over the required image area.
To demonstrate the differences between a simple lens and a quality (well-designed) lens we can
substitute the “normal” lens from a 35mm SLR camera and repeat the above tests. (If you don’t
happen to have such a lens handy, simply follow this discussion -- that’s easier anyway).
Using a “quality” lens, we can observe several things. First, the image formed is now sharper and
covers an area larger than the diameter of the lens itself. Also, the “off-axis” lens defects (known
as “aberrations”) are now well-controlled. And, finally, the image formed is basically “Plan,” that
is flat -- the image is in-focus over most of its area.
Now, of all these optical aberrations the one most relevant to microscopy is “spherical aberration,”
because it alone can be significantly affected by factors external to the microscope, as we will soon
see. (The others are mostly controlled by the microscope’s optics and design.)

2.2 Optics and the Microscope

Now that some understanding of a “Real World” lens has been gained, we can begin to extend this
knowledge specifically to cover microscopes.
As you likely recall, the two principal optical elements of a microscope are the “objective”
(positioned near the object under study) and the “ eyepiece” (near where the user’s eye is
positioned, of course). In the “Real World” it is the objective that mainly determines the potential
performance of the instrument (when properly used, of course) and everything else is essentially
secondary.
However, this is not to suggest that everything else can simply be ignored. For the objective to
perform at its best it must be operated under those conditions for which it was designed. Failing
to do this can result in significant loss of image quality, with the extent of this degradation
somewhat depending upon the choice of objective -- all objectives are not equally-sensitive, as we
will see.

I-24
Understanding the fundamental factors that effect the image, the most appropriate choice of the
optics to be used and their proper application, is what “Real World” microscopy is all about!
Now, this is not as difficult as it may sound at first. Many experienced users perform these tasks
almost automatically, simply as a matter of habit, and of extended experience in the practice of
“good” microscopy. But, to do these things effectively the user must first understand just what the
various considerations are that effect image performance, and how these considerations relate to
those “aberrations” we just observed, as well as a few other relevant factors.
For the sake of simplicity (at least, for the moment) we will assume that all else is just as it should
be with the microscope and only concern ourselves with the objective. We can then move forward
and begin to consider some of the additional factors.
The microscope objective typically consists of one or more lenses arranged so as to create an
enlarged image of the object under study. This resulting “intermediate image” is generated at a
very specific location within the microscope body, so as to have an exact, predefined position
relative to the eyepiece. (The eyepiece further magnifies this image and conveys it to the eye.)
Now, the intermediate image may or may not be “fully-corrected.” In a few modern optical systems
it is, but in most, and especially older systems, it is not. In the past, many of the optical corrections
necessary for the best image were accomplished in the objective, but, because of the technical
limitations of the day, additional corrections had to be performed in the eyepiece. Because of this,
the objective and the eyepiece formed a complementary pairing, each depending upon certain
characteristics of the other in order for the complete “ system” to form the best possible image.
However, given the evolutionary nature of modern optical technology (and lens design) the
partitioning of these corrections, as well as their precise degree, would vary not only between the
various manufacturers but also between successive generations of optics from any one of them.
Consequent, ensuring that the objective and eyepiece being are properly “matched” is one more
consideration in achieving the best possible image quality. Using mismatched optics is quite likely
to result in substandard results. (Some simple methods for determining whether or not the
current eyepiece “matching” is acceptable will be covered in later sections.)

2.3 Microscope Objectives

At this point we can begin to consider the most important optical parts of a microscope, the
objectives and the eyepieces. Although these normally are intended to function in “matched pairs,”
as discussed above, for now we will examine them separately in order to better understand the
factors that govern their performance. Such understanding should allow the user to make better
(e.g., “more informed”) choices in terms of their selection and use for specific applications.
Microscope objectives are made in a wide variety of shapes and sizes, may be intended for any
number of specific uses (from general use to the very exacting and limited), and carry a wide
variety of confusing and often misleading names. In addition, they vary in terms of even such basic
characteristics as mechanical fittings (e.g., mounting threads) and whether or not they feature such
niceties as retractable (“spring-loaded”) tips.

I-25
The maker’s literature is typically the best source of accurate information on any particular line
of objectives (or eyepieces), but specifications may be sparse for offerings from the less-known
makers, and even wholly absent for many “generic” optics.

The vast range of parameters may be summarized (for the most common ones) as follows:

Objective Characteristic Typical Choices Available

Magnification As low as 0.5x, up to 160x, or more.
(but more typically, 4x to 100x.)
NA* Nearly anything, up to about 1.40 max.
Field Characteristics Normal, Semi-Plan (or, Flatfield),
or, Plan. (Other names may be used.)
Optical Corrections Achromat, Fluorite, Apochromat,
with or without a prefix (e.g, Plan.)
Coverglass Requirement 0 (NC, or NCG), 0.17mm, 1.2mm,
(up to a maximum of about 2.0mm.)
Immersion Type None (“dry”), Oil, Water, Glycerin, etc.
Special Type LWD, NCG, UV, Phase Contrast, etc.
Working Distance** Varies with Magnification, NA and Type.
(As little as 0.10mm for some types.)
Intended Use Transmitted Light, Reflected Light, DIC,
or any number of special uses.
Maximum Field Size Unspecified (common) to FN28.***
Tube Length Requirement 160mm, 170mm, 215mm,
also “Infinity”, and others.
Mechanical (mounting threads) RMS (0.8" ,or, “English standard”.)
M24, M25, M26, M27 metric.
Cost Low . . . to Very, Very High!

* NA = “Numerical Aperture,” as is discussed in some detail below.

** “Working Distance” is the space between the coverglass and the
front surface of the objective. (This distance, plus the “Coverglass
Thickness” is the Object Distance, from the front of the objective.)
*** FN = Field Number, in mm., as discussed below under Eyepieces.

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Now, while all of this may begin to seem a bit bewildering, many of these characteristics are on
little concern in everyday use and may safely be ignored. For example, once an objective has been
fitted onto a microscope the specifics of the mounting threads automatically cease to be of any real
concern. The matter of “Tube Length” is a similar issue. In fact, many of the more difficult-to-
understand characteristics only come into play when the selection of a new objective for an
existing instrument is being contemplated -- or when actually changing the optics on the
microscope.
The situation is also complicated by the fact that there is little standardization of the terms used
by different makers to describe these characteristics. Furthermore, markings on the objectives vary
from almost none to full compliance with the most recent DIN standards (where there are so many
markings that finding space for them all may become a problem!). For older objectives, there is
the additional problem that different makers may have used nearly identical marking to indicate
very different things, and/or very different markings for essentially the same things . . .
Nevertheless, a few of these characteristics (or, “parameters”) are usually of considerable
importance in everyday use. These should be understood, and for the most part the rest can be
safely ignored (at least until the occasion to purchase new equipment arises.)
The most common (and “critical”) objective parameters may be listed as follows:

Objective Parameter Application Significance

Magnification (“__x”) Determines size of area viewed.
NA (“x / “, or “NA “) Determines level of detail visible.
Coverglass Requirement Can affect image quality if not
and/or Immersion Type proper for objective being used.
(typ. “0.17" or “oil”) (See Section 2.5 -- Critical Factors.)

Of somewhat lesser importance are considerations such as, Intended Use, Maximum Field Size,
Working Distance and Optical Corrections, all of which may influence the final image but
ordinarily have relatively little to do with its actual quality (e.g., the observable level of detail).
Now, at first it might seem that, since the objective’s NA largely determines the maximum level
of detail in the final image that “the more the better” – but things are not quite simple. As we will
discover shortly, the higher the NA the more sensitive the objective becomes to any deviations from
the exact conditions required for its peak performance.
Thus, for any given magnification, an objective with a higher NA must be used more carefully than
one with a more moderate NA. In addition, the higher NA is usually accompanied by a higher
overall level of optical correction, as well as a higher price. Most lab microscopes are simply
unsuited for use with high-performance objectives, primarily due to condenser and illuminator
limitations (as will be covered later).

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2.4 Microscope Eyepieces
Having gained some understanding of microscope objectives, we can now begin to expand our
knowledge with some understanding of eyepieces. As the name clearly suggests, the eyepiece is
the optical element that conveys the “intermediate image” formed by the objective (within the body
of the microscope) to the user’s eye. In this process the eyepiece may also impart certain predefined
corrections to the image, as determined by the optics’ designer. Inasmuch as any such corrections
are often specific to a limited group of objectives, it is always important to be sure that any
eyepieces used are correct (or at least suitable) for the accompanying objectives.
As with objectives (but even worse), eyepieces seem to have a nomenclature that varies from one
maker to the next, and often even within the product groupings of a single maker. As an attempt
to clarify this situation, some generally respected terms are listed below, along with their most
common usage and meanings:

Eyepiece Characteristic Typical Application or Meaning

Magnification (or, “__x”)
Field Number (or, “FN__”)
Huygens (H, or unmarked)
Compensating “C “ or “K “)
(also, “Kompens”).
Periplan (Leitz trademark)
KompensPlan (or, Kpl)
Widefield (WF, or W...)
Super-widefield (SWF)
High-eyepoint (HEP)
Rating as if used as a hand-held magnifier.
(Typically ranges from 5x to 20x, or more.)
Indicates the size of the “intermediate image”
(actual, in mm.) which will be seen by the user.
(Often unmarked, but normally 16 to 18mm.)
Older-type, simple, low-cost eyepiece having a
narrow field, low eyepoint. (FN = 14 to 16.)
An eyepiece which applies significant optical
corrections to the intermediate image.
An improved design Compensating eyepiece
offering a flattened field. (FN = 16 to 18.)
The Zeiss answer to the Periplan, but having
slightly different corrections. (FN = 18 to 20.)
A better design offering a somewhat greater
actual “Field-of-View”. (FN typ. 18 to 20mm.)
(Good ones exhibit low distortion to the edge
of the visible field -- cheap ones don’t.)
A special eyepiece with a larger-than-normal
barrel diameter. (FN may be 24 to 26+ mm.)
Must be used with suitable objectives.
An eyepiece having an oversize eyelens and
suitable for use by eyeglass wearers.

I-28
Obviously, the two most important eyepiece parameters for the average user are basically the
Magnification and the Field Number. In addition, if the user wears eyeglasses, they may require
a high-eyepoint type. Beyond these, of the many parameters which may be assigned to an eyepiece
the two which cause the most concern and the greatest complications are “color compensation” and
another parameter, the “field curvature” (often somewhat misleadingly labelled, “Plan”).
In the discussions of objective parameters it was mentioned that frequently some additional optical
corrections were necessary in a microscope, beyond just those applied in the objective. To be more
specific, these corrections are most frequently applied in the eyepiece (except in some “infinity”
type systems), and are most commonly the two parameters just mentioned. For many objectives,
as there are only so many things that can be done optically in such a limited space, it is often
necessary to apply corrections for what is termed, “Chromatic Difference of Magnification” (or,
“CDM”). This is an intentional error in the image formed by the objective, where the size of the
details will vary slightly (and in a precisely predictable manner) with both the color of the light and
the distance from the center of the image. It is this condition that is corrected by the “color
compensation” in the eyepiece.
Historically, it has been preferable to make this type of correction via the eyepiece rather than in
the objective itself, and so there are many, many objectives in use today which function based on
this premise. Unfortunately, the precise level of compensation required to exactly balance out this
“residual color” in the image varies considerably between makers, and even between some of the
various objective types from the same maker! Consequent, the best starting point for “matching”
between objectives and eyepieces is usually the manufacturer’s recommendations, typically found
in their published literature. (See Section 2.6 -- The “Mix-n-Match” Game.)
But for older optics, and those from lesser-known makers, this information may simply be
unavailable. Also, frequently it is necessary (or desirable) to operate an objective from one maker
with eyepieces made by a different maker. In such cases the user’s skill in properly “matching” the
two different elements will come into play.
The simplest test to apply is just a critical inspection of the microscope image. What one is looking
for here is evidence of “color error,” as typically seen on the inner and outer edges of object details
which are both in-focus and fairly near the edge of the visual field. With most objectives any color
errors will be revealed by pairings of subtle red-orange and violet color fringes on such edges,
with one color consistently seen on one edge and the opposite color consistently seen on the
opposite edge. Such color fringes within the microscope field serve to indicate the degree of
balance achieved by the objective-eyepiece combination. When this “balance” is ideal, or nearly
so, then there are virtually no such color fringes to be seen on the object!
Objective-eyepiece matching problems exist when these unwanted “color errors” become apparent
in the image. And when they are found, a certain amount of “detective work” may be needed to
isolate their cause and determine a proper remedy. This remedial process begins with an additional,
simple observation.
With many older-design eyepieces there will be a thin, telltale color band along the very edge of
the field, as the eyepiece magnifies its own field diaphragm. Since this magnification occurs
without any contribution from the objective, any “false color” seen here will indicate (usually, but
not always) what sort of color compensation the eyepiece is applying to the image.

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The basic rule, here is: “if the band is blue (or there is none), then the eyepiece is most likely non-
compensating. But, if the band is yellow or orange, then the eyepiece is almost certainly a
“compensating” type.
Note that it is not essential to install eyepieces on the microscope just to perform this test.
Eyepieces can easily be checked for a color band simply by holding them to the eye normally and
viewing the light reflected from a sheet of white paper, or even diffuse light from an outside
window. To a large extent, the strength of the color band seen along the edge of the eyepiece field
will correspond to the strength of the color correction being applied by that eyepiece.

NOTE: The user should also be aware that there are some compensating eyepieces made
which carefully conceal this color band (notably, Olympus WHK’s) in order to produce
a more aesthetically-pleasing, totally color-free final image. These eyepieces eliminate the
typical yellow-orange color band around the edge of the field which is normally seen with
other types of compensating eyepieces. Although such eyepieces will defy the simple
eyepiece test just explained, the table below will still apply.

Armed with this information about the eyepiece type, the user can then proceed to analyze the
typical image color faults and apply an appropriate remedy, as follows:

Image Fault Observed Most Likely Cause Usual Remedy

Objects near field edge show:
Orange fringes on outside and
Blue-violet fringes the inside.
Blue-violet fringes outside and
Orange fringes on the inside.
Minimal or No color fringes.
--------------------------->
“Under-compensation”
“Over-compensation.”
Correct compensation!
Use eyepiece(s) with:
More compensation.
Less compensation,
or No compensation
Change nothing!

Common Compensating-type eyepieces:

Zeiss Kpl and Cpl types (the Kpl’s are very strongly compensating).
Leitz Periplan types (strongly compensating and field-flattening).
Nikon (older) HKW-series eyepieces.
Olympus (older) WF and Bi-WF series/(new) WK and WKH series.
Bausch & Lomb (B&L) “ Hyperplane” or “Compensating” eyepieces.
Any eyepieces marked, “ Comp., Compensating, or Kompens”.
Common Non-compensating-type eyepieces:
Nikon (newer) CF- and CFW-series eyepieces.
Most “ ordinary” AO and B&L eyepieces. Many “infinity scope” eyepieces.
Most generic “DIN” eyepieces (especially inexpensive ones.)

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2.5 Image Formation -- Critical Factors
Under “ideal” conditions, both the objective and the eyepiece (properly matched) will be
functioning optimally, precisely as their designers had intended, and the various aberrations which
detract from the ideal image will be at a minimum, and thus the best possible image should
normally be obtained. However, (in the “Real World”) there are additional, “external” factors
which may disturb this perfect state-of-affairs and thus may cause an inferior image. Fortunately,
these are limited in number and are nearly always at least somewhat under the users control.
This brings us back to the matter of “Spherical Aberration,” as discussed above. This is perhaps
the single most important external factor in many situations and can easily undo any otherwise
perfect setup. It is the lack of appreciation for the effects of this aberration, as well as its causes,
which results in unsatisfactory results for many users. And, should a problem of this nature arise,
these same users are the ones least capable of identifying the underlying causes and of taking
proper remedial action. Yet when this aberration is kept under control, the rest of the parameters
affecting the image can usually be addressed almost as a matter of rote procedure.
Now, spherical aberration may be defined as the inherent tendency for a lens to form an imperfect
image, where the light rays passing through the center of the lens and those passing through the
outside areas do not reach focus at exactly the same location. This situation, when uncorrected,
results in an image that is composed of a mixture of in-focus and out-of-focus light rays.
Under these conditions both image contrast and “apparent sharpness” suffer. However, this
aberration is not too difficult to correct in the design of an optical system and it is this need for
“correction” that accounts, at least partly, for the use of multiple “elements” in the construction
of modern high-performance optics (especially objectives).
Unfortunately, the real difficulty here is that complete correction can be achieved only for a
specific set of optical conditions. In the case of the microscope, these conditions include not only
having the correct spacing (or, “tube length”) between the objective and the eyepiece, but in also
maintaining a specific set of conditions external to the objective as well.
These “external conditions” are primarily concerned with the exact nature of the optical path from
the object to the front surface of the objective, including such factors as the thickness and the
“Refractive Index” of the mountant and the coverglass. In fact, these conditions are so important
to the creation of an optimal image that they are often inscribed on the barrels of most modern
objectives!
Now, the “Refractive Index” is just a number (a measurement, actually) which indicates the
relative ability of a substance to bend light rays. Nearly every solid substance that can pass light
with reasonable efficiency (is, “transparent”) is likely to have a refractive index measurement.
Naturally, this measurement is extended to include many of the liquids commonly used in
microscopy, too.
The table on the following page lists the “Refractive Index” values for a number of common
materials encountered in microscopy.

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For reference, some of the more common Refractive Index values are:

Optical Material Refractive Index

Air 1.000
Water 1.333 (at 20 deg. C.)
Glycerin 1.42-1.47 (varies with water content)
Glycerin Jelly 1.454 (mountant -- formulas vary)
Diatoms 1.434 (Silex / silicon shells)
Mineral Oil 1.48 (“Heavy” type mineral oil)
Mountants 1.51-1.53 (depends on specific type).
Immersion Oil 1.515 (typ., at 20 deg. C.)
Cover Glass 1.52 (typ.-- plastic types may differ)
Canada Balsam 1.535 (dried mountant)
Cellulose 1.54 (typ. plant cells)
Styrax 1.58 (typ. used for diatoms)
Zrax (mountant) 1.72 (typ. used for diatoms)
Pleurax 1.77 (typ. used for diatoms)

From this brief listing it is obvious that the Refractive Index of Immersion Oil is nearly identical
to that of a Cover Glass. Thus, this special oil is essentially the equivalent of “liquid glass!”
However, also note that there is a significant difference between these substances and both air and
water. The importance of these differences in microscopy will now be explained . . .
Returning to the matter of “Spherical Aberration,” we find that the principal cause for the
appearance of this problem in a normally perfect microscope optical system is simply a failure to
respect the proper operating conditions for the objective. Now, not all objectives are equally
susceptible to this problem. And, somewhat surprisingly, its severity is not directly related to the
magnification of the objective – instead, it is related to its resolving power, or, “NA”.
What this means to the microscope user is simply that some objectives will be much more sensitive
to this problem than others, but that this sensitivity is also rather predictable. And since the primary
cause of this problem is well understood, its control can be undertaken fairly easily.
In microscopy, the principal cause of excess spherical aberration is simply the failure of the user
to respect the operating requirements for the objective in use, or, more specifically, to ignore the
effects of a refractive index error in the object-to-objective light path. And what all this boils down
to for most objectives is primarily the matter of “Coverglass Thickness.” (See table on next page.)
As you may observe in the earlier table, the Refractive Index for air is quite different from that of
optical glass. And one of the parameters considered in the design of precision microscope
objectives is exactly how much glass (or its equivalent) will be placed in the path between the
object and the very front surface of the objective. This is because, from a strictly optical
standpoint, it makes no difference where this glass is positioned in that path (as long as it’s a flat
piece or layer). And, on that basis, this glass may simply be considered as part of the first lens of
the objective!

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Thus, as the thickness of the coverglass is changed, it is effectively the equivalent of altering the
thickness of the glass in the front lens of the objective -- and that is why it can be so important to
good image quality to have a coverglass of the correct thickness (especially when using “high-NA”
non-immersion objectives), as shown in the following table.
This sensitivity to coverglass thickness exists, to some degree, for all “dry” objectives of moderate
or higher NA. And, the higher the NA, the greater the sensitivity, as shown here:

Objective NA Coverglass Thickness* Range**

0.25 (or less) 0.00 to 0.50mm. n/a
0.40 0.10 to 0.25mm. +/- 0.07mm.
0.50 0.12 to 0.22mm. +/- 0.05mm.
0.60 0.14 to 0.20mm +/- 0.03mm.
0.70 0.15 to 0.19mm +/- 0.02mm.
0.80 0.16 to 0.18mm. +/- 0.01mm.
0.90 0.165 to 0.175mm +/-0.005mm.
0.95 0.167 to 0.173mm. +/-0.003mm.

* Based on an objective designed for standard 0.170 mm coverglass.

** vs. 0.170 mm. Std. No.1 coverglasses are 0.10 to 0.15mm thick,
while the No. 1-1/2 types are generally 0.13 to 0.19mm thick.

This is the reason why many high-NA “dry” objectives are equipped with a “correction”
(adjustment) collar -- to permit the adjustment of their internal optics so as to minimize the effects
of coverglass thickness deviations from the nominal. Failing this, the image quality will deteriorate,
the extent of which is dependent upon the specific “effective coverglass thickness.”

“Why would anyone make such problematic lenses?”, you may ask . . . The answer to this
is that for some applications, a high-NA objective is required for reasons other than
resolving power. The best example of this is a technique called, “Epi-fluorescence,” where
a light beam (usually Ultraviolet, or “UV” light) is projected down through the objective
onto a specimen which has been treated with a special, fluorescing substance. The
excitation of this substance by this light causes it to glow, albeit, weakly -- often very, very
weakly. Now, the more intense the beam is, the brighter this glow (or, “fluorescence”) will
be, and the easier it will be to detect. Thus, for such applications, a high-NA objective is
usually desired, if not essential, as the high-NA results in a brighter image.

One additional concern here is the existence of any mountant between the underside of the
coverglass and the portion of the object under study. In this instance, when objects of any real
thickness are examined it may be found that image quality varies as the objective is focused on
different parts of the object. This is because some parts of the object may be embedded more
deeply in the mountant than others and, as the objective is focused on these different parts, it must
operate through optical (mountant) layers of different thicknesses. The “effective coverglass
thickness” includes any mountant between the underside of the coverglass and the part of the
object that is in focus.

I-33
With this in mind, note that one way to reduce the effects of coverglass thickness error is just to
lower the “ effective NA” of the objective. For brightfield microscopy, this means simply closing
the condenser iris a bit -- enough to restore the lost image contrast without sacrificing resolution!

NOTE: To allow for the presence of at least some mountant in the image path, the
“No. 1" coverglasses were standardized at a nominal thickness of 0.013mm,
instead of the expected 0.17mm thickness specified for most objectives. However,
for more precise work, where the object may be mounted directly in contact with
the underside of the coverglass, a “No. 1-1/2" coverglass thickness was created,
having the correct 0.17mm nominal thickness.

Based on the above table it is readily apparent that at least some image degradation is to be
expected even when using the “basic” 40x/0.65 objective, and that the extent of this degradation
will be determined by the actual “effective” coverglass thickness (coverglass + mountant).
However, all this is not meant to imply that this problem affects only “dry” objectives. Immersion-
types can also be affected by a similar problem (although this is much less common).
In the case of “dry” objectives we are obviously concerned with the “Effective Coverglass
Thickness.” But in the case of “immersion-type” objectives we need to be concerned with
something which might be termed, the “effective immersion index.” Here there are different factors
to consider. The reasons for this are twofold. First, we need to use the correct immersion fluid --
immersion oil in the case of oil-immersion objectives and water (or glycerine) in the case of those
types. Additionally, we need to be concerned about the coverglass and mountant as well, especially
in the case where the objective relies on a non-oil immersion fluid.
The main reason for this unexpected concern is the very high NA of most immersion-type
objectives. While “dry” objectives are limited (by optical physics) to a NA of less than 1.00, most
immersion types have no such restriction and may have a NA of 1.30, 1.35, or even higher. For
such high NA’s especially, the old problem of spherical aberration reemerges.
One situation where this problem can occur, and is rarely foreseen, is in the case where old, thick
diatom mounts are being examined with an oil-immersion objective. Here the actual coverglass
thickness is not a concern, as its refractive Index is closely matched to that of the immersion oil.
However, many of these mounts were purposely made with very high-index mountants, and some
of these can have a Refractive index of 1.75, or even higher. So, in such cases, it is conceivable
that any appreciable thickness of such a high-index material might well result in increased
spherical aberration and thus degrade the image. The irony of this situation is that these high-index
mountants were developed and used specifically to enhance the visibility of the diatom markings,
yet if misused (e.g., in excessive thickness) could easily result in a significant deterioration of the
overall image!
Another instance where this problem can arise with an immersion objective is when the objective
is used with an inappropriate immersion medium. For example, many older objectives were made
which used Glycerin (R.I. = 1.42) as their immersion medium. Using such objectives with standard
immersion oil (R.I. = 1.52) introduces a significant error (in terms of refractive index). The extent
to which this error causes visible deterioration of the image depends on the thickness of the layer

I-34
of the improper fluid. Thus, in cases where the objective’s “Working Distance” is small, such
substitutions may still result in an acceptable image. But with objectives having a long working
distance, greater loss of image quality should be expected.
An even more insidious situation may occur with a Water Immersion objective used in water, but
without the proper coverglass allowance. Many of these objectives were designed for use without
a coverglass and so their images can suffer significantly if a coverglass is inadvertently introduced
into the light path (simply out of habit, usually) when they are used. Others were designed for use
with a special, matching “dipping cover” (basically a tiny coverglass built into a holder which
fitted over the tip of the objective). These objectives normally require the use of either a
coverglass or their original covers, otherwise their images will be degraded.
The unfortunate part of all this is that very few of these special objectives are marked to indicate
their exact requirements. So, all too frequently some experimentation is needed to discover what
is actually needed for the best image. And this leads us to the next topic for discussion . . .

2.6 The “Mix-n-Match” Game

One very tricky area of microscopy is the desire (or even legitimate need) to “mix-n-match”
objectives of one maker with eyepieces from another maker. This may happen, for example, when
a particular objective is needed (or wanted) but the instrument it is destined for is already equipped
with expensive eyepieces from a different maker. (It makes little sense to use expensive
objectives with cheap eyepieces -- there are reasons why really good eyepieces are expensive
and the cheap ones are simply not in the same league, no matter what sellers may claim!)
In such scenarios, the number of parameters to be considered can be overwhelming and most users
barely have any idea as to where to begin.
Fortunately, there are some “Rules-of-thumb” which may be applied to simplify things:
(1) “Plan” objectives complicate the situation greatly. If you are getting a new objective
just because it is “Plan,” and try to use it with another maker’s eyepiece you are
likely to be disappointed. “Plan” objectives are designed with a specific “image
curvature” and this is typically “flattened” by corrections made in the matching
eyepiece. Since the different makers usually choose to do this in different fashions,
actually achieving a “Plan” field in “mix-n-match” is largely a matter of luck.
However, this is not to say it cannot be done. If the objective has other special
features the user needs, and the “Plan” characteristic is truly secondary, then success
is more likely. Chances of an acceptable result also improve if the eyepieces used
do not have extra wide fields.
(2) “Oddball” objectives complicate the situation greatly. Optics from lesser-known
makers frequently use old or dated technology, thus reducing the likelihood of a
match with more modern equipment. This also applies to things like objectives
with very special design parameters, such as using quartz coverglass. This is
okay if you have the proper covers, for example, but not if you plan to use just
ordinary “stuff.” When the makers mark their optics with special requirements,
they aren’t kidding!

I-35
(3) “High-end” objectives complicate the situation greatly. Dying to have that new
“super” Plan-Apo? Think twice . . . The higher the level of correction an objective
has, the more exacting the demands it places on the eyepiece. You can buy the
objective, but don’t expect it to perform nearly as well as it could with the correct
eyepiece. If you do not have the correct eyepiece, you might be better served with
a slightly “lesser” objective. In fact, you might not see any difference between them
when using your present eyepieces!
(4) “Achromats” are usually easy to match. These are the least demanding of all
objectives, and therefore place the least demands on the eyepieces. For many
applications, an Achromat is all that is needed. And, while you can still get into
trouble with these (see the following section), your chances of success are higher
than with more demanding objective types. Just be aware that compensation
requirements can very widely among achromats, even if they are from the same
maker (and the same “series”).
(5) “Tube Length” is not a major problem (usually). Here we fall into the “fallacy trap.”
For many objectives, the “160mm” and “170mm” types are nearly identical! Why?
Because “tube length” is not the critical parameter -- the distance from the “
objective seat” to the “intermediate image is actually what counts, but no one
specifies this. This is one way the makers have of “enforcing” brand loyalty. (See
Section 2.7 for more on this.)
(6) You cannot mix “infinity” and “finite” optics. This is a real “No-No!” Infinity optics
are specially designed such that they require an extra lens (the “tube lens”) within
the body of the microscope in order to form a proper image. With no tube lens
present you may not get any image at all. Worse, each maker has their own
“formula” for such a lens, and this makes the “mix-n-match” game a real nightmare
when played with “infinity” optics.
(7) Parfocal distance is not a killer, just a pain. In years past there were many very good
special-purpose objectives made, for which there may not be any equivalent modern
replacements. A significant problem with many of these is that they are of the
“short barrel” type and thus cannot be brought into focus on many newer scopes --
because they are simply too short. Fortunately, there are several remedies available
for this situation. Leitz made an adapter (“PLEZY”) specifically to permit their older
“37mm” objectives to be used on their newer (45mm, or “DIN”) instruments. Also,
simple fixed-length adapters can be used, 10mm being a common length. Finally,
there is an adjustable adapter (the “DIN-Adapter”).
(8) “Widefield Eyepieces” complicate the situation. The wider the eyepiece field (FN)
the more difficult it will be to get a successful “match,” if you’re insistent on having
a Plan image. The wider the eyepiece field, the more of the “curved” intermediate
image it will have to contend with, and the less likely a random match will yield a
successful result. On the other hand, if the very edges of the field-of-view are not
that important to you, perhaps because of your application or your specimen
characteristics, then the eyepieces are less of a concern.

I-36
(9) Photography complicates the situation greatly. Because the human eye automatically
re-focuses as it travels over the field-of-view, what appears to be perfectly Plan may
not truly be so. Makers exploit this “accommodation” capability of the eye so they
can relax their design requirements and thus minimize their costs. So, in most cases,
if you want to get a really Plan photo you will have to use the objective makers
designated eyepieces to get it.
(10) Avoid older optics -- “the newer the better”. Technology marches on! In optics as
in most other areas. Thus, older optics are quite frequently “ bettered” by their
modern counterparts. Many older optics use “uncoated” or “single-layer coated”
lenses, as well as older design and assembly technologies. Such optics can be very
sensitive to illumination conditions and image glare is frequently a problem.. If your
scope lacks a proper “Field Iris” you may well have difficulties getting satisfactory
images.

NOTE: As a consequence of this, that 20-year old “Plan Apo” you’re eyeing may
not even be as good as the newest Plan Achromat! Factor in other things, like aging
of the glass and the internal cements, the higher quality of today’s multi-layer lens
coatings, etc., and you just might want to rethink that aging lens . . . .

(11) “Color-free” is no free lunch. Nikon’s finite (“160mm”) “Color Free” (or, ”CF”)
optics were ground-breaking when they were introduced in the late 1970's, or so.
With CF optics all of the chromatic corrections were made in the objective, making
them incompatible with nearly every other maker’s equipment!
This is especially true for the Plan members of the CF-series due to the resulting
field curvature corrections in the CF-series eyepieces. Still, there are some members
of the CF family which can be used quite successfully with other makers eyepieces,
most notably the excellent CF Fluors (e.g., with many Olympus eyepieces).
(12) Zeiss is “not-so-nice”? Being quite an old design (1950s) the Zeiss “Kpl” eyepieces
seem to have far more color compensation than does almost anything else (except
perhaps the Leitz Periplans). Although they are truly excellent optically, they do not
necessarily mate all that well with other makers objectives. But Zeiss’ lower-cost
“Cpl” eyepieces are less-fully compensating, and so might be a much better choice
if you insist on mixing other makers’ optics on your Zeiss microscope.
Also, you should be aware that the Zeiss (and Leitz) design philosophies seem to
result in making as much color-correction as possible in the eyepiece, whereas other
makers (most notably Nikon) prefer to do it in the objective. Thus, mixing optics
between Nikon and these other makers usually leads to rather poor results. In
such extreme cases it may simply be best to avoid attempts at “mix-n-
match.”

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2.7 The Great “170 vs. 160mm” Debate
It is not uncommon for users playing “Mix-n-Match” to become concerned about the classic “170
vs. 160mm tube length” problem. Actually, this is all rather meaningless. And here’s why . . .
This great concern stems primarily from the fact that for many, many years Leitz (mostly) made
their microscopes with a “170mm” mechanical tube length, while other manufacturers used a
“160mm” tube length. Thus, Leitz was viewed as unique, and incompatible. Their objectives were
all marked “170"(mm) and users feared that awful things could happen if they tried to use them
on their 160mm microscope.. But, in actuality (in the “Real World”) this is NOT about objectives
at all, but simply about the eyepieces!
It seems that the early Leitz “Periplan” eyepieces, had an extremely long barrel (compared to other
designs). Their microscopes featured extra-long eyetubes, which kept the eyepiece ends from
hitting the prisms in a binocular body, and the extra eyepiece length interfered with their use on
other maker’s microscopes. Since the eyepiece tubes had been extended by 10mm, these
instruments were known as “170mm tube length” types and the objectives to be used on these
stands were then marked as 170mm types (“170/ ”). And so, the “170mm vs. 160mm” confusion
inevitably began.
However, the objectives are not the problem. Relative to the rear of the objective (or, actually the
nosepiece face) the location of the intermediate image produced by these objectives is almost
exactly the same as that produced by most 160mm objectives. Thus, “170mm” and “160mm”
objectives are interchangeable -- Leitz adjusted for the 170/160mm tube lengths in their eyepieces!
This was shown clearly when Leitz changed to the “160mm tube length” standard in the late
1970's. Leitz simply provided 10mm spacers so that their “170mm” eyepieces could be used in
the newer (shorter) “160mm” scopes -- and the optics stayed the same. Users could then freely
“mix-n-match” their Leitz 170mm and 160mm objectives on their new “160mm” stands.
If you measure Leitz optics, you will find that the older Leitz 170mm eyepiece designs have a
“shoulder height”* that differs, by exactly 10mm, from the newer (“160mm”) types. And that’s
the heart of the situation, and the big “170mm vs.160mm Debate” secret -- it’s all in the eyepieces!

* NOTE: The “shoulder height” measurement reflects the location of the eyepiece “field
stop” vs. the upper end of the microscope tube. It defines the expected location of the
intermediate image produced by the objective. Thus, “170mm” eyepieces should only be
used on a 170mm stand, or a “160mm” stand, with 10mm eyepiece spacers added. (To use
“160mm” eyepieces on a “170mm” stand you would either have to cut 10mm off the
eyetubes, or live with the error.)

Now, if you could measure the optics, you would find that most of the ”160mm” objectives made
by major makers in the past 20-30 years (e.g., Nikon, Olympus, Zeiss, etc., plus most DIN’s) use
the same “optical tube length,” the same as Leitz, within approximately +/- 1mm.!

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2.8 Parfocal Distance
Just when you thought your “Mix-n-Match” problems were over, along comes “Parfocality.”
Parfocality is the mechanical condition where different objectives can be rotated into position with
the nosepiece and remain nearly in focus. (They are then said to be “parfocal.”) This is a great
convenience to the user and so most users expect their objectives to have this property.
Now, to be “parfocal” the objectives all have the same “Parfocal Distance.” This means that, when
used as designed, the distance from the nosepiece face (the objective “seat”) to the object will be
the same, within a small margin of error. Nearly always, the objectives from any one maker’s series
will be parfocal. The few exceptions to this are almost always either objectives intended for special
uses (e.g: thick covers), or those with “adjustable” features of some sort.
All this immediately creates a problem in the “Mix-n-Match” environment since, over the years,
there have been many different “Parfocal Distance” standards. Many users will recognize this as
the “Short-barrel vs. Long-barrel” situation. However, in the “Real World” there are thousands of
objectives available in the “short barrel” category, and for these the parfocal distance can range
anywhere from about 33m to roughly 43mm -- with each maker clearly doing his own “thing” back
when they were made, as shown below:
Typical Parfocal Distances for common “transmitted light” (“Biological”) objectives:

Manufacturer Objective Parfocal Distance

American Optical* 160 mm 34 mm.(Achromats)
160 mm 35 mm (Apochromats)
Bausch & Lomb 160 mm (old) 35.5mm (may vary.)
(1970s...) 160 mm (Japan) 43 mm. (typ.)
Leitz (1950s...) 170 mm (short) 37 mm.
(1970s...) 170 mm (long) 45 mm.
(1980s...) 160 mm (long) 45 mm (DIN)
Nikon (1970s...) 160 mm (short) 33.5 mm
(1970s...) 160 mm (“ M”) 33.5 mm
(1980s...) 160 mm (new) 45 mm (DIN)
(1980s...) 215 mm (“ M”) 45 mm
Olympus (70s...) 160 mm (short) 36.5 mm.
(1970s...) 160 mm (“ M”) 43 mm.
(1980s...) 160 mm (long) 45 mm (DIN)
Zeiss (East G.) 160 mm (old) 33 mm. (“Jena”/LOMO)
(West G.) 160 mm (new) 45 mm (DIN)

* Also known as “AO” and “AO/Spencer”. Switched to “infinity” optics in the 1960's.
NOTE: The dates shown in the above table are only approximate.

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The term, “Long barrel” is commonly applied to those objectives meeting the new “DIN” standard,
which specifies a 45mm Parfocal Distance (and “160mm Tube Length). Thus, most new
microscopes, being equipped with DIN-style objectives from the factory, only focus to about
43mm, or so (approx. 45mm, with a little extra margin added).
Obviously, if your microscope won’t focus any closer than 43mm, you potentially have a real
problem in trying to use older objectives -- a situation that the various makers no doubt well
understand. The DIN standard effectively prevents many users from accessing their stockpiles of
the older objectives, simply because they can’t be focused on the newer scopes! (This is one
reason why these objectives are now so available used at such low prices -- normally, “DIN-
only” type scopes cannot use them!)
However, all is not lost -- there are some potential “solutions” (or, “fixes”) available . . .
One common solution is simply to use an older scope. Many very good scopes predate the “DIN”
era and were capable of working with “Short Barrel” objectives. In fact, most of these scopes were
equipped with such objectives when new. (Examples include: Leitz Ortholux-I, Nikon S-series,
Olympus original CH models and their BH-series scopes.)
Zeiss [West Germany] switched to the 45mm standard back in the 1950s. (The only Zeiss “Short
Barrel” scopes were made by Zeiss Jena [East Germany] under various names.) So the only Zeiss
scopes that will work (unmodified) with the older objectives are the “Zeiss Jena” scopes.
Another solution, should you prefer to use them on your new “ DIN-only” scope is a small adapter
made by Leitz, called “PLEZY.” This is a 37mm to 45mm objective adapter with a built-in lens
to compensate for the tube length error created by adding the extra 8mm. These work well but are
not precisely parfocal, and they are not cheap either. However, the additional tube length does not
disturb image quality until the objective NA is above about 1.1 or so. Thus, for a standard objective
on a scope with an Abbe condenser, a vintage Leitz “PLEZY” may be an excellent solution!
Some users opt for simple mechanical “extenders,” 10mm being a common “off-the-shelf” length.
This works well with short-barrel objectives having parfocal distances as short as 34mm and so
is a popular solution for users of the older optics. A thin washer or loop of wire can be added to
adjust for the 1mm or so error and can thus make such a setup nearly parfocal with DIN optics. It
is also possible to shorten these adapters a bit (usually on a lathe) to permit their use with
objectives in the 36-37mm range, thus preventing the “adapted” objective from hitting the slide
when mixed with DIN objectives on a nosepiece.
Finally, until recently, there was another option available, the so-called “DIN-Adapter.” This
device was essentially a “variable extender,” would support short-barrel objectives from 33.5mm
to over 37mm, and, as it was adjustable, could be set to make the “adapted” objective exactly
parfocal with DIN objectives. Its design also included features for minimizing glare, often a
problem with the simple extenders. (Sadly, the MicrAP “DIN-Adapter” is not currently being
manufactured.)

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2.9 “Infinity” Optical Systems
The growing proliferation of microscopes with “Infinity”type optical systems in the past few years
has probably led to more misunderstanding and confusion in microscopy that anything else. What
this is all about is very simple -- “infinity-focused” optics can be “split” without causing the sorts
of image distortions that occur in conventional (“finite” or, 160 /170mm) systems due to the added
“tube length.”
In a conventional microscope, if you have a good high-NA objective that is functioning ideally,
and then just lengthen the body tube by a couple of inches, you will find that no amount of
focusing can restore the original image quality -- it’s been lost. This is because standard optical
systems (“finite tube length”) have strict optical requirements, and, on a microscope, the “tube
length” is one of them. (Technically, aberrations are designed to be at their minimum when the lens
system is functioning at its proper tube length. The higher the NA, the more critical this becomes.)
Because of this problem, any accessory optics which are inserted into the body tube (e.g., via an
Intermediate Tube) must also include some special optics to correct for the effects of the extended
tube length. (This is why many of the older “intermediate-type” accessories are marked “1.25x”
or so -- because of just such extra internal optics).
However, “infinity” systems have no such restriction. Their optical performance remains largely
unchanged regardless of the “tube length” of the system (within reason, of course). This
characteristic of “infinity” systems then allows all sorts of accessory optics to be inserted into the
optical path with little concern about them degrading the image.
This, of course is a great benefit to the manufacturers, who can now invent all sorts of exotic
setups to sell to their users. Examples include some exotic combinations previously not possible
with the older “conventional”optical systems, such as “Epi-fluorescence + Nomarski DIC,” now
fairly popular in some specialized research applications. You can’t get much more exotic (or
expensive) than that!
The way this (“infinity”) is accomplished is fairly straightforward -- an additional lens is
introduced into the optical path at a specific distance in front of the eyepieces. This lens, called a
“tube lens,” is responsible for much of the final image formation in the instrument, and often
includes some of the functions formerly accomplished by the eyepieces, such as color-
compensation and field-flattening.
Now, it appears that some makers have designed their tube lens specifically to allow them to keep
using their old eyepieces, although other makers (Nikon and Olympus, for example) have designed
entirely new eyepieces. (And, once again, every manufacturer has their own ideas about the
specifications for this lens, and that makes switching optics between different makers’ “infinity”
systems very, very difficult -- so we now have an entirely new way of enforcing “brand loyalty.”)
An unfortunate side effect of all this is that the new “infinity” objectives now frequently exhibit
much shorter working distances than their “ finite” counterparts. Many oil immersion objectives
are now specified with a “Working Distance” of around 0.10mm, whereas the older designs were
often close to twice that. This is a problem because it limits just how deeply the lens can be
focused into a mounted specimen (below the underside of the coverglass).

I-41
On the other hand, advancing optical technology (plus one more lens to work with, the Tube Lens)
has provided many “infinity” systems with absolutely superb image quality, particularly in such
areas as Phase Contrast, where the objectives are especially complex.
Finally, it should be mentioned that, in addition to providing optical “correction,” such as “field
flattening” and/or “color compensation,” the tube lens on an infinity-type microscope also very
much affects the actual magnification!
This is because the tube lens of the microscope, in conjunction with the eyepieces, serves to form
a powerful telescope, and it is through this “telescope” that we essentially view the image formed
by the objective. Thus, the more powerful this telescope is, the greater the magnification.
So critical is this relationship between the tube lens and the eyepieces that this lens is rarely made
as a part of the microscope, but rather is almost always made a part of the eyetube unit.

NOTE: One advantage of this route is that it often allows users to “upgrade” their older
stands to use the newer infinity optics by simply fitting a new eyetube unit. Olympus is
one example of this, they made a simple adapter which permitted their UIS-system
“infinity” tubes to be used on their “standard” series microscopes. Thus, users could
upgrade their existing Olympus microscopes to use the very latest Olympus optics at quite
reasonable cost!

However, another crucial point with all of this is that, although “infinity” objectives from one
manufacturer may fit another maker’s microscope (mechanically), they also might not function
well at all, even to the point of providing totally incorrect magnification. Just as an example, we
can use the “old” Leitz infinity system, which used objectives with standard (RMS) threads.

Because of the physical length of the Leitz microscopes of that era, and the fact that, in that
particular system, the “tube lens” was positioned in the nosepiece, the “tube lens focal length” was
made unusually “long” (approximately 240mm). This results in the following situation . . .

Most of today’s infinity objectives are designed to work with a tube lens in the 150-200mm range
(depending upon the maker). So, using one of those “old” Leitz objectives on a newer infinity
microscope (non-Leitz) can result in a very large magnification error -- in this case, perhaps only
60%, or so, of the marked value! On the other hand, using a newer infinity objective (such as one
of the popular AO Plan achromats) on an older “Leitz’ infinity microscope can result in excessive
magnification -- in this case approximately 150% of the marked value.

So, as you can see from this example, the switch to “infinity” optics has created a whole new level
of compatibility problems if you try to play the “mix-n-match” game!

NOTE: If you should have access to any of these various items it may be fun to
experiment (why not . . . ?) Just be aware that the results you achieve can be
very different from what you might normally have expected.

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2.10 “Real World” Choices
Now that you have a general understanding of microscope objectives and eyepieces, it is time to
take a look at the “Real World.”
First, we shall deal with eyepieces. Most microscopes are equipped with “10x” eyepieces and these
represent 95% or more of the eyepieces actually used. For most applications these are at least an
acceptable choice, and generally are also the best choice. They tend offer the best overall
compromise between image sharpness, field-of-view and viewing comfort. Also, if the user must
wear eyeglasses with the microscope, there may be very few alternatives.
Still, there are alternatives -- typically 8x and 15x-16x eyepieces. The “8x” types have two main
uses: (1) to restore 10x total magnification for accessories which have a 1.25x magnification factor
(“intermediate-tube” type accessories), and (b) to improve apparent sharpness and contrast with
high magnification (e.g., 100x, or so) objectives. These eyepieces are often made as “high-
eyepoint” types and so are compatible with eyeglasses.
The 15x or16x eyepieces, on the other hand, are used when it is desirable to obtain a higher image
magnification with a given objective -- most typically with higher-NA objectives. While these are
occasionally produced in high-eyepoint versions, the actual eyepoints still tend to be lower than
with the 10x and 8x types, so eyeglass compatibility may be marginal. On low-cost versions, the
eyepoint may be very close to the eyelens, making the use of such eyepieces uncomfortable. Also,
even for the best versions, the “field-of-view” tends to be limited, typically only 2/3 that of the 10x
eyepiece.
Because of these problems, a few makers provide 12.5x eyepieces. These can offer a number of
desirable features, including high-eyepoint, full “ field-of-view,” and increased magnification.
Many users prefer these to the more powerful types listed above, for obvious reasons. Also, the
combination of full “field-of-view” and increased magnification gives a wider “viewing angle” --
often described as a “super widefield view”! This, plus the fact that the modest (25%)
magnification increase makes constant viewing of fine detail less taxing on the eyes, tends to make
these eyepieces the “favorites” of those users lucky enough to have access to them. Unfortunately,
all these features come at a cost, these 12.5x eyepieces tend to be rather expensive -- often nearly
twice the cost of the 10x equivalents.
In any event, the rules for “matching” discussed earlier in this chapter still apply. While those
superb Zeiss 12.5x Kpl eyepieces may be highly desirable, they would be a very poor match for
your Nikon CF-series objectives, for example.
So this, naturally, bring us to the subject of objectives. Unfortunately, these tend to be far more
complicated than eyepieces. And it also brings us back to face the issue of “upgrading”!
Unlike eyepieces, the selection of objectives is as least partly governed by the application -- the
selection of objects to be viewed as well as the viewing methods. For example, a 10x eyepiece will
work just as well with a 4x objective as with a 40x, but these objectives are used for very different
types of objects. So, understanding the potential application requirements is a proper first step in
choosing an objective. Once the application has been decided, then the real selection process can
begin.

I-43
The factors that generally govern microscope objective selection may be listed as follows:

Magnification Compatibility
Resolution (NA) Working Distance
Optical refinement (e.g., Apo, Plan) Cost

While many new users begin by assuming that Magnification is all-important, rarely is. In fact,
the actual magnification may differ considerably from that marked on the objective, by 10% or
more. Thus, a “40x” objective may actually have a magnification of 36x to 44x, and this range may
be even wider for low-cost imported types. Further, somewhat surprisingly, it is usually the
cheaper objectives that have the higher magnification, while the “better” objectives are more often
lower than marked.
Why is this so? Because it is often easier (cheaper) to increase the magnification just a bit in an
effort to make the visual field appear flatter than to make a true “Plan” objective. However, when
you do have a good “Plan” design, then makers often reduce the magnification a bit to make the
image appear a bit sharper, and to increase both the “field-of-view” and working distance.
As a good “rule-of-thumb,” the higher the magnification the smaller the “field-of-view” and the
shorter the working distance. (Note: Object Field = Eyepiece Field /Objective Magnification.)
In terms of resolution (NA), this is often limited by the microscope. There is little to be gained
from adding a NA 1.40 objective to an ordinary lab microscope, for example, unless all the
supporting elements are also present (quality illuminator, corrected condenser, precision fine-
focus, etc.) In most cases a high-quality Plan Achromat (NA 1.25 or 1.30) would be a more
appropriate choice, and would likely prove much easier to use.
This brings us to the matter of the diminishing performance differences between the latest Plan
Achromat (or Plan Fluor) objectives and the actual “Plan Apos.” While, historically, these
differences may have been considerable, today it can often be difficult to distinguish between these
simply on the basis of image quality. And, although the price differences have remained
significant, the performance differences generally have not. (Two series of Plan Achromat
objectives that offer “near Plan Apo” performance are the Olympus “S-Plan Achromat” series and
Nikon “CFN Plan Achromat” series.)
In terms of “newer” versus “older” objectives, except for specialized types were no modern
equivalents are available, the newer objectives are nearly always superior. This is due not only to
improved designs, but superior glass types and improved manufacturing techniques. Thus, that old
Zeiss Planachromat (a very good objective) can be easily challenged by an inexpensive Nikon CF-
E lens! (Assuming proper eyepiece matching, of course.)
Thus, while it once was fairly common for users to “upgrade” their standard “Lab” scope with
expensive, older optics, this practice may no longer offer the benefits that it once did. In fact, many
of the “standard” objectives found on the latest “unknown maker” scopes are actually good enough
that simply replacing them (as was once the practice) may no longer provide any significant
improvement at all.
So, with all that in mind, let’s now turn to one remaining critical issue, the Illumination . . .

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3. Microscope Illumination
3.1 “Real World” Microscope Illumination
Obviously, when using a microscope it is necessary have some form of illumination in order to be
able to see anything. However, the choice of illumination (and its setup) can have significant
effects on the optical performance of the instrument.
Years ago (and even today, with “Vintage” microscopes) illumination was provided by from an
external light source and redirected up into the microscope optical system via a gimbaled mirror.
This system, originally developed for use with daylight and kerosene lamps, has now largely been
superseded by built-in “illuminators” of varying degrees of sophistication. These, in turn, may
utilize a variety of light sources, from standard lamps to low-voltage, compact-filament Halogen
lamps, to compact fluorescent lamps, and even to battery-powered LEDs.
Needless to say, some of these illumination systems work better than others, in terms of the level
of microscope optical performance they will support. Some are barely suited for low-power, non-
critical observations, while others are capable of supporting the most demanding research-type
applications. Why such a huge difference in capabilities? Isn’t one “light” as “good” as another?
The answers to these questions require a bit of background, but such understanding can make a
very significant difference in the users level of success in microscopy. So let’s begin . . .
The simplest form of illuminator may be considered the “diffuse” type. If one reads very old books
on microscopy they may find that the advice of that day, for the “best” source of illumination, was
a sunlit white cloud in the sky! However, for an instrument equipped with a mirror, in a time when
the only reasonable alternative may have been a fluid-burning lamp wick, this advice made some
sense. The suggested white cloud was in fact a distant, “diffuse” light source.
Today, a close approximation to this form of illumination may be had from a desk lamp with a
large light bulb. Ordinary light bulbs are not well suited inasmuch as they typically exhibit a “hot
spot” (a limited, central area of excessive brightness). Still, there are some “decorator” type bulbs
which are comparatively large, spherical, and have a very white, dense diffusing surface. These
can make excellent light sources for some uses (e.g., for vintage scopes!), and at very low cost. The
main reason for their unexpected effectiveness is that the light from these lamps satisfies some of
the more important criteria for good microscope illumination: it is adequately bright, uniform, and
“distant-diffuse.”
Unfortunately, it is far more common to find the “cheap” forms of diffuse illuminators, the little
light boxes which may be freestanding but are more typically either built-in or plugged-into the
microscope itself. These consist of little more than a boxed lamp and a piece of “ground glass”
(which acts as a “diffuser”). For most of these gizmos, the lamps are too small and too weak, and
the diffusing surface, unaccompanied by any optics, is located much too close to the microscope.
These devices are barely useful as they fail to satisfy the above criteria: their light is too dim
(particularly with the typical dark-blue glass filter in place), it is non-uniform (even with the added
“diffuser”, they usually exhibit uneven illumination), and the light is too highly diffused, often
resulting in “glare.”

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So, now we have two seemingly similar light sources, one of which is perfectly acceptable and one
which is clearly not. Why is this so? What are the specific factors that make this difference? This
is where we begin the process of really understanding microscope illumination!
To obtain a decent image with a microscope you must have adequate image brightness. This
translates into a light source which is “adequately bright” (not too dim, but not excessively bright
either). Cheap illuminators usually fail in this respect -- their lamps are just too small and too weak
anything but low-power work.
The next issue is one of uniformity; the light source should be capable of providing a uniform
background for the microscopic object, one that is very even in appearance. Here again, the cheap
illuminators often fail. Lacking any useful optics and with their lamp filaments typically too close
to the ground glass surface, they almost always exhibit a noticeable “hot spot.” And this is a
problem when attempting low-power work -- so cheap illuminators don’t work well for that either!
This brings us to the matter of the characteristics of the light rays reaching the microscope.
In microscopy it is highly desirable to have a well-controlled source of illumination, not one where
light is just “spraying” all over the place. Light from such a controlled source is said to be
“collimated” -- that is, the light rays are all headed almost exactly in the same direction. (A better
way to think of this might be that the light rays are traveling in a tight “ bundle” and spread out
very little.) The optical effect is similar to shadows on a sunny day, which have high contrast and
are sharply defined -- just as we would like our microscope images to be!
However, with a “diffuse” source, the effect is more like an overcast day, where all the shadows
are “grayish,” lacking in contrast, and with fuzzy edges (poor sharpness). This is because “highly-
diffuse” illumination is “uncontrolled” light, with the light rays basically arriving at the object
from random directions. Now, since the object is illuminated from many directions at once, this
makes the edges less distinct and lowers the “contrast” (difference between light and dark areas).
Furthermore, when the illumination is not well-controlled, it may take unwanted paths through the
optics and the microscope itself. Problems of this sort simply require using a different (better-
designed) illuminator.
Diffusion is often used to obscure defects in a light source (such as a visible filament) but this
process also results in significant light loss (due to “scattering”), so the light is also made less
bright -- another problem.. Excessive “diffusion” is just a “cheap fix” for other problems!
Now, the main difference between cheap illuminators and the simpler desk lamp arrangement
described earlier (with a proper light bulb) is that the light in the desk lamp is “distant” -- that is,
it lies at a significant distance from the microscope. And there lies a very important lesson about
microscope illumination. For a lamp that is quite close to the microscope, the light is “spraying”
all over, totally uncontrolled. But for a diffuse lamp placed at an adequate distance (1-2ft.), the
situation is actually quite different. Here the direction of the light rays can only vary by a
comparatively small amount (by the time they reach the microscope). Technically, the light from
a distant diffuse source is nearly “collimated” (as mentioned above), which is highly desirable –
but, it is also principle upon which an excellent illumination system may be built. All that is
needed now are some additional optics to allow proper control of the light when it enters the
microscope!

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3.2 Illumination and the Condenser
Before we discuss more advanced methods of illumination, we need to understand a bit about the
proper functioning of the microscope condenser. (Even if your current microscope lacks one, you
should still read this section, if only to better understand what you’re missing. For those readers
not already somewhat familiar with the condenser, it’s that “funny looking thing” which moves up
and down underneath the stage. Many low-cost and “School” type scopes lack one.)
Now, many users believe that the condenser is there to somehow “concentrate” the light for a
brighter image, but this is not so. The true purpose of the condenser is very different -- the
Condenser actually makes the microscope work -- that is, it makes the microscope able to reveal
fine detail !!!
“Whoa! -- how is that possible?” you might ask . . . Just read on . . .
As you may recall from the above discussions of the microscope objective (or possibly from other
reading), objectives are characterized by their “NA,” which is an indicator of their ability to
resolve fine detail -- the higher the NA the better they are at showing fine object detail. The
problem with this is that in order to actually utilize the full NA of the objective it is absolutely
necessary to fill the “full aperture” of the objective evenly with light. Simply directing light up into
the objective will not accomplish this (except for basic, low-power objectives). What is actually
required is a well-controlled “cone of illumination” -- and this is where the condenser comes in.
The condenser is a powerful (usually) lens system, working something like a magnifier, but in
reverse. Here the illuminating light enters the larger end of the condenser and becomes focused
down to a small area around the object on the microscope. Because the condenser can be moved
up and down (again, usually) it may be adjusted to focus an image of a distant object (such as the
light source) directly on the object (or, sometimes, even elsewhere).
In fact, this method of illumination, where an image of the light source is focused on the object
being studied, was quite common about 100 or so years ago -- when the principal sources of
illumination were oil lamps featuring very wide wicks. With these, the image of the light source
on the object satisfied the conditions for a uniform, distant, diffuse light source. (Of course, the
brightness might have been an issue, but that was the state of the technology way back then.)
This form of controlled illumination was termed, “Critical” (or, “Abbe-Nelson”) Illumination and
was widely used even into the 20th Century (often by using very special electric lamps). For
strictly visual use it was, in its day, considered unsurpassed. However, for photography through
the microscope, where rather bright light sources were required (due to the slow “speed” of the
glass photographic plates of that time) something different was required. In order to secure an
acceptable form of controlled illumination, utilizing the early, rather crude electric lamps (or even
arc-light sources) the “Kohler’s Method” of Illumination was developed.
These days, if you glance through nearly any book on microscopy (above the juvenile level) you
are likely to find several pages devoted to touting “Kohler Illumination.” In fact, it is now quite
rare to find “Critical Illumination” even mentioned in a current book, except very briefly. Now this
is quite an ironic situation inasmuch as more than 95% of the available microscopes simply cannot
actually provide true “Kohler” Illumination!

I-47
Most of these microscopes actually use a form of Critical Illumination and even for those that
claim to provide Kohler, most actually only provide a crude approximation of it, which is really
just another form of Critical Illumination! The debate over the relative merits of these two
competing forms of illumination raged for many years, with “Kohler” proponents eventually
proclaiming victory. But that debate ended nearly a century ago, when microscopes were designed
very differently, and when optical technology was still relatively primitive. So, perhaps now is a
good time to reexamine the underlying issues of this debate, in the “light” of current technology.
But first, will we need to examine these two methods and understand their goals, their advantages
and their possible weaknesses.

3.3 Kohler vs. Critical Illumination

Many texts on microscopy feature fancy depictions of light rays streaming through a microscope,
attempting to convey some understanding of Kohler’s “method of illumination.” Most of these
seem to fail at this task, not be cause of the quality of these drawings, but perhaps because these
proponents of Kohler do not actually understand the method themselves and are simply parroting
what they were once taught. The basic problem with all this is simply that the Kohler method is
now become “oversold, ” to the exclusion of some viable alternatives.
What August Kohler accomplished with his 1893 illumination method was a technique for
addressing the primary illumination problems of that day: (a) that most microscopes were equipped
with the very basic (and mediocre) Abbe condenser, (b) that intense illumination was required for
photography through the microscope (which necessitated the use of rather small light sources), (c)
that high image contrast was required (again, for photography), and (d) that a uniform background
illumination of the object was required for a decent-looking image.
Now, the Kohler method accomplishes all of this rather admirably. The basic problem is that this
method requires the use of a rather elaborate light source. Historically, this was not a problem as
most microscopes required a separate light source anyway, so the user was free to choose which
one best fulfilled his requirements. But today, that flexibility has largely been lost (except for some
of the costlier research-type microscopes which can accept various light sources). This simple fact,
that the Kohler method requires a sophisticated illuminator, is generally not mentioned, nor is it
properly depicted in those fancy drawings, which typically show only a simplified light source.
What is required to implement strict (“true”) Kohler illumination is a light source with the
following special characteristics:
(a) A compact-filament lamp, preferably one that is “centerable” to the optical axis of
the light source. This is to minimize subsequent distortion of the filament image.
(b) A well-corrected optical system (not just a simple “condenser” lens), which can be
focused. This is required to project an accurate image of the lamp filament into
the front aperture (“ front focal plane”) of the microscope condenser.

(c) An absence of any diffusing gimmicks in the lamps optical path. (These were a later
addition, to compensate for a lack of lamp adjustability and for lower-quality optics.)

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Very few microscopes are available today which can satisfy Kohler’s requirements. And for those
models that have such equipment available, it is rarely provided in the “standard” form of the
instrument but instead must be specially ordered. (However, this has not always been the case. For
example, Nikon produced several models in the 1970's, the S-Kt, S-Ke, and L-Ke stands, which
all featured excellent Kohler illuminators as standard equipment, and these were competitively
priced. Unfortunately, such instruments are an exception and today these vintage stands are highly
prized primarily because of their excellent illuminators.)
One additional complication with the Kohler method is the complexity of its setup. Here, one must
execute a sequence of not-so-simple tasks in order to ensure complete and proper establishment
of Kohler’s conditions. These steps are summarized in the following table . . .
The classic setup steps for establishing “true” Kohler Illumination are as follows:
(1) The lamp “Field Iris” is opened and the lamp filament image is sharply focused
on the “ front focal plane” of the microscope condenser. (If necessary, the lamp
filament may also need to be “centered” within the light source and Field Iris.)
(2) The object is selected and brought into sharp focus in the microscope.
(3) The lamp Field Iris is closed down somewhat and then the microscope condenser
is adjusted so as to bring the image of the Field Iris into focus in the object plane.
(The Object and Field Iris are now “superimposed” on one another, both in focus.)
(4) The light source and/or condenser are adjusted so as to center the image of the
Field Iris, which is then opened to just slightly beyond the visual field limits.

As you can now see from this listing, there are few microscopes available which permit these sorts
of adjustments to be made. (The main reason for this is that most users are simply unwilling to go
through all these required steps and seem well-satisfied with simpler illumination systems.)
So now, you might ask, “If Kohler’s Method is so wonderful, why is that so very many experienced
users just don’t want to be bothered with it?” (I warned you that it was oversold . . .)
And, you might also ask, “If these users are willing to settle for less, just what are they using and
just how “acceptable,” or, “inferior” can it be? (Ah, now there’s the real question!)
Many of the better, albeit simplified, microscopes of today feature what is actually only a rough
approximation of Kohler Illumination. Such instruments are characterized by simplified light
sources (usually built-in) and a greatly simplified setup procedure, typically as follows:
(1) The microscope Field Iris (Stop) is opened. (No Field Stop = No true “Kohler”!)
(2) The object is selected and brought into sharp focus in the microscope.
(3) The Field Iris is closed somewhat and brought into focus in the object plane
by adjusting the condenser focus. (Same as with “true” Kohler.)
(4) The microscope setup is then adjusted so as to center the image of the Field Iris,
which is then opened to just slightly beyond the visual field limits. (Same, again.)

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However, note that in this implementation there are no lamp focusing, centering, or other lamp
adjustments required. Usually, these adjustments are “factory set,” to simplify things, but they are
also typically completely unavailable to the user. So the user must accept whatever settings the
factory technicians have “pre-set.” And while lamp replacement in a proper Kohler light source
can be a tedious process (because of the need to re-center the lamp), this too has been “simplified.”
Most lamps today are pre-centered and may simply be plugged into their socket, and usually there
are no centering adjustments provided. So another opportunity to “optimize” things has been lost.
Now, “just how is all this wonderful ‘simplification’ possible?” you might ask . . .
Once again, just read on . . .
The answer is, simple -- the makers simply added some “diffusion” to their light source, thus
converting it back into the “diffuse” type of source we first discussed, and this eliminated the need
for all those extra time-consuming steps. (Although, those “extra” steps were the very things that
made Kohler Illumination work so well in the first place, but now the illumination has been
“simplified.”)
The plain fact is that now, with this ubiquitous “ simplified Kohler” method upon us, we have
essentially reverted to using another form of “Critical Illumination,” or, at best, a hybrid of these
two, formerly competing methods (Kohler vs. Critical). So, now might be a good time to review
the basic differences between these methods. We can then decide just what it is that we actually
have with this “Simplified Kohler” illumination, and perhaps how to best utilize it . . .

3.4 “Simplified” Kohler Illumination

In spite of all the various diagrams often used to “clarify” these two methods, the basic difference
is very simple; in Critical illumination an image of the source is focused in the “object plane,”
whereas with Kohler Illumination the source image is relayed to the objective’s “rear focal plane.”
Thus, with Critical you view the source and the object at the same time (superimposed), while with
true Kohler you can only view the source when viewing the rear of the objective.
However, with “simplified Kohler” Illumination, there’s essentially nothing to view in either
location as there is never any “real” image of the source. So, to resolve this dilemma we need to
slightly revise our definitions of the Critical Illumination method to account for this.
As might recall from earlier discussion, Critical Illumination was originally developed to utilize
a broad, glowing flame as the source. Now, much unlike a lamp filament, such an object is both
an “unstructured” source and an efficient “Lambertian source” (simply, a “uniform” radiator). So,
despite the problems, like coloration, lack of intensity, and flickering (motion), these old sources
were optically nearly ideal for visual use (at least, theoretically).
However, with the advent of the electric light, all this changed, as the broad flame was now
superseded by a slender, glowing filament; the former “unstructured” source was replaced with a
new source that had an appreciable “ structure” to it. This made early electric lights unsuited for
use with the Critical method as such attempts merely resulted in an annoying image of the electric
filament superimposed on the object. (And, so, this is why Kohler’s Method was developed.)

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But, the electric light sources were “improved,” initially perhaps simply by the expedient of
grinding a “diffusing spot” on the glass shell of the lamp, and later perhaps by the introduction of
a ground- or opal-glass diffuser in front of the lamp. All of these methods had the effect of
minimizing (or even eliminating) the annoying light source “structure,” but at a cost of some loss
of light intensity (due to light scattering by the diffuser), and, in the case of ground-glass, of
introducing a new (albeit, rather fine) structure, the surface of the ground glass (still a problem on
some scopes).
Subsequent development improved not only the electric lamps but also the efficiency of the diffuser
arrangement. It was learned that adding a simple “collector lens” in front of the diffuser would
improve the efficiency of the illuminator, for example. Also, if an iris diaphragm was positioned
immediately after the diffuser, then the apparent size of the light source could be altered, much as
is done with Kohler Illumination to control “glare.” So, at this point, we now have “Modern
Critical Illumination” -- the lamp flame has been replaced by an electric light source, a simple
collector lens (often with either a separate or integrated diffusing surface) and a Field Iris.
If, at this stage of the argument, you are starting to suspect that this “modern” illuminator
configuration is suspiciously like “simplified Kohler” you are exactly correct -- the two methods
are very nearly identical!
So, now the distinction between “true” Kohler and Critical Illumination becomes very simple:
Kohler Illumination requires a highly-corrected lens system in the Illuminator, which
is used to project an sharp image of the lamp filament into the front of the microscope
condenser. This results in an image of the lamp filament in the rear of the objective.
Critical Illumination uses a simple lens system in the Illuminator, often with a diffuser.
A Field Iris may be added (similar to Kohler) to control the apparent size of the source.
The microscope condenser focuses an image of the apparent source onto the object plane.
(There is NO image of any lamp filament, although there may be one of the Field Iris).
Both methods, properly configured, will provide acceptable illumination with the usual
Abbe-type microscope condenser, but the Kohler method will be brighter as there is
properly no diffuser in the light path to introduce “scattering” (or, “diffusion”) losses.
For either method, improved results with high-NA objectives may be obtained by
replacing the basic Abbe condenser with one that is more fully-corrected -- and this is the
subject of the following two Sections on Condensers . . .

NOTE: It seems worth mentioning, once again, that the basic “Abbe” condenser is actually quite
limited in terms of the “Effective NA” that it can support, regardless of how it may have been
marked by the maker. The marked NA numbers, 1.20, 1.25, 1.30, etc. are simply an indication of
the maximum angle of the “ light cone” exiting the condenser, and say nothing about how
effectively that cone is focused. The Abbe performs poorly for objective NA’s above about 0.7 and
can seldom support an effective NA much greater than about 0.9 (dry), or 1.1 (using “oil
immersion”) – perhaps why these condensers are so rarely “oiled” to the slide (there is so little to
be gained by that.) This “Abbe” problem is discussed further in the following sections. (You may
also wish to refer back to the table on the top of page 14, which details such effects.)

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3.5 Higher-Performance Condensers
The Abbe Illuminator (as it was originally named) as become one of the most widely used optical
devices in history. Roughly 95% of all “real” microscopes are equipped with one, with mainly the
lowest-cost student type scopes (having either no condenser or just a simple built-in “stage lens”)
excluded. It was introduced in the early 1870's as Carl Zeiss (Jena, Germany) began serious mass-
production of microscopes. For the Continental users of the day, its ability to illuminate a wide
field (for use with low- and medium-power objectives) was a great attraction. (Reportedly, it was
never actually intended for such use, merely as a demonstration device for Abbe’s theories on
microscope resolution and image formation.)
However, it was cheap-to-make, and it satisfied some of the basic requirements of the day, so it
went into mass production, over a hundred years ago, and is still with us today. But, as standard
equipment on almost every “real” microscope, users should understand its workings, as well as its
limitations, so they can best utilize it -- and also possibly recognize then their needs might be better
served by replacing it with something better!
The basic lens design of the standard Abbe consists of two lenses, a large, lower lens and a smaller,
“hemispherical” top lens. Together, these provide a maximum NA of about 1.2 (although some
makers stretch this a bit and mark their Abbe’s as high as NA 1.30). But, regardless of how they
are marked, inherent optical defects in the Abbe design limit its “effective NA” significantly.
The “standard” two-lens Abbe is an “uncorrected” optical design -- there are no additional
elements within it which remove (or cancel) the basic limitations of its simple lenses. Thus it must
exhibit some serious optical defects, the two most obvious of which are “chromatic” (color) and
“spherical” (focal-error) aberrations. Anyone who has used one of these has likely noticed the
strong “false-color” fringes which appear inside the opening of the source. Field Iris (unless the
scope lacks one of these). These are evidence of it significant “chromatic aberration.”
Less obvious are the effects of “spherical aberration,” although these may easily be observed if one
knows just where to look -- in this case, the rear focal plane of the objective. Now, spherical
aberration is a non-linear phenomena, rapidly becoming more serious as the lens aperture (NA) is
increased. Because of this, the Abbe only functions reasonably well up into the middle of its NA
range, roughly up to about NA 0.65 or so. (This makes it well suited for use with the common
40x/0.65 objective, while its wide field coverage allows it to be used with powers as low as about
4x, which explains its popularity.) However, for NA’s much above about NA 0.70 the Abbe’s
effectiveness becomes increasingly limited as it simply cannot properly (“evenly”) illuminate the
rear aperture of the objective. And this occurs whether it is used “dry” or “ oiled” to the slide.
As most microscopes are equipped with a 100x “oil immersion” objective (NA 1.25, or so), this
makes a reasonable vehicle with which to demonstrate the Abbe’s limitations. If one sets up the
microscope normally, using the 100x objective, and then removes one eyepiece (so as to better
view the rear of the objective) one should see the Abbe’s iris as it is opened and closed. However,
as the iris is opened toward the maximum aperture of the objective, it may be noticed that it no
longer retains its normal appearance, and instead may “break up” into annular rings or “beads”.
In essence, the iris has no effective control over the objective aperture at these higher NA’s, when
this occurs, and thus cannot contribute to the proper control of any “glare” arising within the
microscope optics.

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But, even worse, it may be noticed that even with the Abbe’s iris fully open, the rear aperture of
the objective is not fully and evenly illuminated. And, this condition cannot be remedied by any
focus position of the Abbe. No matter how it’s positioned, the necessary “even illumination”
cannot be achieved. This means that the objective cannot function at its full, rated NA! In order
to work at its rated NA, the objective rear aperture must be fully and evenly illuminated.
Otherwise there is a loss of usable NA, roughly similar to closing down the condenser iris.
It may also be observed that similar results are obtained whether the Abbe is used “dry” or is in
oil contact with the underside of the slide (using “immersion oil”). Mainly, all that results from
using immersion oil here is that two glare-inducing “air-to-glass” surfaces are eliminated. The
effective NA of the 100x objective is improved only marginally, if at all -- which is probably why
most users don’t bother to use immersion oil (on their Abbe condenser) but simply use it “dry.”
Now, none of this is meant to imply that the Abbe design is worthless, merely that it does have its
“real world” limitations and that the user should be aware of these and learn to respect them. The
inability of the Abbe design to properly illuminate the aperture of the 100x objective is the primary
reason that most workers can never really achieve maximum results from these lenses. And, as
nearly all microscopes suffer this handicap (the Abbe), most workers never have access to a scope
that is capable of effectively using such objectives.
Fortunately, all that is required is to replace the simple Abbe with a “corrected” condenser to
achieve a dramatic improvement in the image! The following table lists some of the more common
condenser types, along with their main characteristics and advantages:

Condenser Type Typ. NA F.L. / W.D.* Advantages

Abbe (2-lens) 1.20 to 1.30 13mm/2mm Very inexpensive, easy-to-use.
Aspheric (2-lens) 1.20 to 1.35 13mm/2mm An “improved” Abbe type.***
Achromatic 0.90 /0.20** 12mm Reduced Chromatic aberration.***
(also 1.25) Suitable for color photography.
Abbe (3-lens) up to 1.40 9mm/1.5mm Basically an “Aplanatic” design.
Aplanatic (3-lens) 1.30 to 1.40 (varies) Reduced Spherical Aberration.
Superseded by Achro-Aplanatic.
Achromatic/ Highly refined optically. Minimal
Aplanatic 1.35 to 1.40 9mm/1.5mm aberrations, when used properly.
Requires centering for best results.
Ultra-Low typ. 0.16 max. 65+ mm. Required for Kohler Illumination
with low-power objectives (1x-4x).

* This column lists both condenser “Focal Length” and “Working Distance”.
** The “ Achromatic” type is now quite commonly made as a “ Swing-out” type,
having a dual NA. -- approx. 0.90 with the top up, and 0.25 with it moved aside.
*** Some of these condensers may also have corrections for Spherical Aberration.

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3.6 Special-Purpose Condensers
There are any number of transmitted-light microscopy applications for which the “standard” types
of condensers are either “less-than-ideal” or wholly unsuited. In addition, there are alternative
contrasting techniques which may be used to increase the visibility of object details or produce
other specialized illumination effects.
The most common of these “non-standard” uses may be summarized as follows:

Macro (and Photomacrography): This involves the use of low-power objectives (1x-4x)
on a standard microscope for the purpose of viewing/photographing extra-large objects,
such as large sections of organs or entire specimens. Ordinary condensers simply are not
usable over such large object areas and special condensers are often required to obtain
uniform illumination.
Darkfield: This method uses a special condenser type which eliminates any direct
illumination. Instead, the object is uniformly side-lighted and appears “glowing” on a near-
black background. This method works very well with small, transparent objects (i.e.,
diatoms, desmids, algae, protozoa, etc.). Standard objectives may be used.
Oblique: In this method the direct lighting is modified so as to illuminate the object at a
side-angle, thus (when properly executed) resulting in a “pseudo-3D” effect. It is an
alternative to Darkfield and standard objectives are always used.
Rheinberg: This is an old but very effective method for producing “color contrast.” It
relies on applying a special color filter to the condenser which results in striking color
differences in the object. As with Oblique, standard objectives are always used.
Phase Contrast: This is a widely-used technique for viewing live, transparent objects.
However, it requires special objectives and a special condenser. Some variation in image
contrast is obtainable by using objectives with different contrast characteristics.
Anoptral Contrast: Results are similar to Phase Contrast but the specifics of this
technique are slightly different. Special phase objectives and condenser are required. Not
widely used as it is currently only supported by one or two manufacturers.
Hoffman Modulation Contrast: This is a patented contrasting method which results in
pseudo-3D images, similar to those obtained with the Oblique method. Although it also
requires special objectives and special condenser, standard objectives may be modified (by
Hoffman) to use this method without interfering with their normal use.
Fluorescence: This is primarily an obsolete technique. It relied upon a darkfield-type condenser
(which was capable of transmitting UV light) so as to excite a fluorescent dye in the object. This
object fluorescence was then observed normally through the microscope, although special filters
were also needed to prevent the UV light from reaching the eyes. It has been almost entirely
replaced by superior Epi-fluorescence.

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Now, it seems only fair to note that many of the condensers cited in the above summary are often
based on the Abbe design. In addition, it is possible (or, in some cases even necessary) for the user
to make minor alterations or additions to a standard Abbe condenser in order to achieve some of
the results listed.

The applicability of the common Abbe condenser design for the above methods is as follows:

Macro: Frequently, the Abbe condenser is manufactured in a “divisible” form. In this case
it is simply a matter of unscrewing the removable top to obtain a low-power condenser of
about NA 0.3, which is nearly ideal for work with 2x - 4x objectives.
Darkfield: This is a favorite amateur modification for the Abbe and there are several
ways to accomplish this. The simplest is called a “Patch Stop” method, where an
“occluding disk” is inserted into the condenser’s filter holder. (These were once provided
with the microscope as standard items, but now must usually be ordered separately.) When
installed, the Abbe is converted to darkfield use and usually works very well with
objectives of up to (and including) 40x/0.65. The very defects that normally limit its
usefulness serve to make it most effective for this use.
Oblique: This conversion is similar to that used for Darkfield, but this time the “stop”
is eccentric. Such “oblique stops” can be made in a wide variety of shapes, and this is a
favorite area of experimentation for many amateurs. Again, this method works best for
objectives up to (and including) 40x/0.65.
Rheinberg: Another method for which the Abbe is well-suited. Here, transparent stops
are made typically using a pair of complementary colors, usually in a concentric
arrangement, but radial arrangements are also used. In use, the central area colors the
object background while the edges of the object are side-lighted in a contrasting color
(somewhat similar to darkfield). This is another favored area for experimentation. And,
again, this method works best for objectives up to 40x/0.65.
Phase Contrast: The special condensers required for this method are frequently simply
an Abbe type with the mechanical addition of a rotating turret. The turret holds a set of
“rings” which are designed to match up with complementary “phase plates” installed in
special objectives. Although the technique is very powerful, it is also very popular and so
the equipment is not priced unreasonably. In addition, this method has been in use for
nearly fifty years now, so there is a considerable amount of used phase contrast equipment
available. However, the restrictions are that the condenser must fit the microscope being
used and the phase objectives must match the condenser.
Hoffman Modulation Contrast: These condensers usually Abbe types, modified in a
manner quire similar to phase contrast condensers but using linear slits, rather than annular
ones. This, if a Hoffman Contrast objective can be obtained, it is not too difficult for the
user to fabricate his own slit, which may then be positioned near the lower focal plane
(iris) of the standard Abbe condenser.

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3.7 Proper Illumination?
One of the best-kept “secrets” of microscopy is the need for verifying the illumination setup!
While many, many texts on the subject of microscopy (yes, even this one!) list step-by-step
procedures for illumination setup, almost none detail methods for verifying that what has just been
done has resulted in a proper and effective configuration for your equipment. This failure is one
of the chief reasons why so many users encounter difficulties in using their equipment and/or seem
to be able to achieve only mediocre (or even unsatisfactory) results. And these failures can hardly
be blamed on the users (or their equipment) when no one has taken the time or made any effort to
explain to them how to detect setup errors or equipment faults and then how to remedy them.
So, perhaps for the first time in history [just kidding . . .] that effort will be made -- here . . .
Now, in the entire microscope there is probably no area less visited nor any more poorly
understood than the rear focal plane of the objective. Yet this is probably the single most critical
point in the entire image-formation chain for it is in this location that the “intermediate image” is
created. (The “intermediate image” doesn’t actually appear here, but this is basically where
it originates.)
Perhaps one of the reasons this point remains so obscure is that viewing it on a routine basis can
become a bit tedious. For the diligent worker, this would entail removing one eyepiece, inserting
and focusing a “phase telescope” (or similar device), observing this location (and possibly making
some adjustments to the instrument and/or its illumination), and then removing the viewing device
and restoring the eyepiece! At least, this would be the common practice -- and it could get tiring!
However, there is an alternative method, one that is easier, quicker and simpler. Also, it does not
require any such costly device as a phase telescope. This alternative method relies on the fact that
in a microscope the rear focal plane of the objective has a “conjugate” image located external to
the instrument. That is, everything that can be seen in the original location will also appear in the
image at this alternative location. But most users are unaware of this “secret” location.
“And, just where is this ‘secret’ location,” you might ask?
That’s easy, it is at the “eyepoint” of the eyepiece -- the one location where all the light rays from
the rear of the objective reappear in their proper optical relationships!

Except for size and any color-compensation which may be applied by the eyepiece, the
image at the eyepoint is exactly like the one seen in the rear of the objective!

However, just to clarify what this is all about, this is absolutely NOT the image of the object that
one normally views. No, this is a different image we are now discussing . . .
Furthermore, this “secret” image cannot be seen directly. When you place your eye at the so-called
“eyepoint” of the eyepiece all you can see is the normal image of the object -- and this is NOT
what we are presently looking for. What we want to see is something very different . . .

I-56
To do that we must understand exactly where the “eyepoint” is and just what must be done to make
this “secret” image visible. (Many books mention the “eyepoint,” but very few explain it.)
Most simply, the “eyepoint” is the precise position above the eyepiece at which the lens of the
users eye must be positioned in order to view the entire microscope field-of-view.
If the user’s eye is too high or too low, then only part of the field may be seen. Fortunately, users
quickly learn to position their eye(s) correctly and so this becomes almost automatic and
instinctive.
Now, demonstrating the location of the eyepoint is fairly easy. With the microscope set up
normally, (using the10x objective) simply take a white card or small sheet of paper and hold it
about 3/4-inch above the eyepiece, such that the eyepiece projects a small circle of light onto the
card’s surface. The exact position of the “ eyepoint” may be found by moving the card to and
away from the eyepiece, very slowly, until the position where this projected circle of light is
smallest is found. This position is the eyepoint of the eyepiece (normally 14-18mm above the end
of the eyepiece, but closer for some older type eyepieces).
Just be aware that the spot is quite tiny, only about 1-2mm in diameter, and that the card must be
positioned quite accurately for this demonstration to succeed. (Fortunately, this method is just for
demonstration purposes. The “real” viewing technique is much, much easier.)
So, if we know where the eyepoint is, and that is where we normally place our eye, why is it that
we never see this “secret” image?
The answer is, that it is not optically possible to see this image with the unaided eye. What we
“see” instead is the “normal” image. However, if you return to the location demonstration just
performed, and inspect that tiny circle of light very carefully, you may notice that it looks an awful
lot like the rear of the objective (just reversed, because the image is now projected). Furthermore,
if you close open and close the condenser iris, while still viewing the projected image, you may
also see that iris movement in the image (but only if the condenser is correctly focused . . .)
If you can find the “eyepoint,” the position at which the spot of light is smallest and sharpest, then
you have accomplished what is necessary -- you now know where the ‘secret’ image is located.
However, if you were unable to see any details within the spot, not to worry -- they can be very
hard to see with the unaided eye -- a problem we shall soon remedy . . .
As you can now acknowledge that seeing much of anything in that tiny spot of light is very
difficult, at the least, and may seem nearly impossible (unless you have excellent eyesight at close
distances) we can assume that you just might be interested in an easier way view this hidden image.

So, here it is . . . simply use a fairly powerful (10x or 15x) hand magnifier!

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3.8 “Verifying” Your Illumination
Once upon a time, Nikon (and probably a few others) one made a neat little device for doing
exactly what we are about to do, but those are now rare and are (sadly) no longer made. Although
the image was a bit smaller than that obtained with a phase telescope, it had the unique advantage
that it functioned “through” the eyepiece (not in place of it) and so it was very quick and easy to
use. we can accomplish very nearly the same thing with a well-chosen hand magnifier. It needs to
be “achromatic,” so that it does not introduce a lot of false color into our observations, but not
much beyond that. It does not need to be super-expensive. Most of the decent-quality imports will
work just well. It needs to have at least a “10x” magnifier rating, although more will make for a
bit larger image to view. Just remember that the higher the magnification, the more difficult it will
be to position and hold steady. So, 10x-14x would be just about right for most users.
Another choice, at least for a beginning, would be to use an old Abbe condenser (there is a certain
irony there!). These can function as roughly a 16x magnifier when used with the eye at the large
lens end. Just be sure to open the iris fully, and to keep your fingers out of it.
Once a suitable magnifier has been obtained it’s time to learn how to apply it to viewing our
‘secret’ image. To position the magnifier correctly, remember that for this use, it must be located
at the “eyepoint” position plus the focal distance of the magnifier -- the focal distance can be found
simply by using the magnifier normally to examine something and noting the distance from the
magnifier to that object. The magnifier should then be positioned above the “eyepoint” (and not
the “eyepiece”) by about that amount.
With the magnifier in approximately the correct position, it may be used to view the ‘secret’ image
-- although this may take a bit of “ trial-and-error” the first few times. (However, this is a skill
quickly and easily learned by most people.) The learning task is made easier if the condenser iris
is closed a bit more than normal, such that the iris leaves “shadow” into the objective aperture.
This provides an easier “ target” for the magnifier and is what we will want to see anyway.
As part of the learning exercise, you may wish to remove the eyepiece and view the rear of the
objective directly a few times, just to compare its appearance with what you see through the
magnifier. Once you believe you are viewing the correct thing, you may try opening and closing
the condenser iris (while still using the magnifier) to verify that you can observe its setting and its
operation. And, after doing this with the 10x objective you may wish to try the 40x.
While all of this may seem like more trouble than its worth right at the moment, remember that this
is just a basic “familiarization” exercise, and that once you “get the hang of it” the entire viewing
process (to verify the illumination setup) should require only a few seconds!
And, after about 5 minutes of this you should very nearly be an expert at it . . .
So, now that we have the special technique learned, just what are we looking for?
That’s easy -- everything that we have already covered under Illumination. Specifically, we are
looking to see an uncolored, uniformly illuminated objective aperture, with the iris set so that it
is just a bit less than the maximum aperture of the objective. In other words, “perfect” Illumination.

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Unfortunately, at least at this stage of things, we are more likely to see one or more defects in the
existing setup. And this is actually good, since it demonstrates the potential value of this viewing
technique -- already, within just a few seconds you can determine whether or not your illumination
setup is satisfactory. (You should feel really good about this because about 95% of all users
couldn’t do that given a week, let alone do it in just a few seconds!)

NOTE: The methods suggested above should be considered as merely an alternative to the
more conventional methods, namely: Direct Viewing or using a Phase Telescope. For
those users requiring “reading glasses” the use of the magnifier method may be easier than
trying to view the rear aperture directly. The important point is that this
viewing/checking should be performed quite frequently, typically with each
magnification (objective) change, if the user is to consistently achieve the best results.

Now, there’s a very easy, reliable way to accomplish nearly the same results . . .
Once you have reached the point where you can typically set up the illumination correctly, and
merely want to “optimize” it, all you have to do is look for the “contrast bump.” This occurs when
the condenser iris has just reached its optimal setting (all else have been set up correctly). If you
begin with the iris wide open, while observing the specimen (in sharp focus) and then slowly close
the condenser iris, you will notice a point where the image contrast suddenly increases -- this may
be a little or a lot, depending on the objective, the specimen, the condenser and the Field Iris. When
you see this “contrast bump” then you are at (or very near) the ideal setting for the condenser iris!
So, “Why reveal this shortcut only now?”, you ask . . .
Because, (1) everything else must be correct (or very nearly so) for this shortcut to be most
effective, and (2) for many specimens you may find that the best condenser iris setting differs from
this “ideal,” depending upon what part of the specimen you are observing ( particularly with larger
stained “sections” where different areas may be stained quite differently). Also, if everything else
is not correct, this shortcut by itself, may very well lead to worse” results if the condenser iris is
closed down too far, to cover-up other setup faults!
Now, even with this shortcut, the previous methods are still quite useful in at least one more area --
setting the Field Iris. If you have an objective which is sensitive to the Field Iris setting, you should
see this when viewing its rear aperture, as follows:
With the Field Iris wide open, and the Condenser Iris closed, so as to reduce the objective’s
effective aperture slightly (or, the “normal” condenser iris setting), you should notice that there
is a bright ring seen outside of the Condenser Iris’ image. This “ring” is due to glare resulting from
the “extra” light allowed into the objective by the wide-open Field Iris, and which is reflected off
the inside of the objective barrel, reducing image contrast. Setting the Field Iris correctly will
almost always eliminate this “glare” ring, (This problem occurs most with older high-NA
objectives.) So, the next steps in this new process are to learn to identify a few more common
illumination problems (using their appearance with this method) and then to learn how to go about
correcting them.

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3.9 Correcting Illumination Faults
Before we begin an in-depth discussion of what we should observe, what it means, and what
actions may be needed as a result of what is seen, perhaps we should consider the selection of an
appropriate viewing technique, one which best suits the user’s particular requirements. The reason
for this is that should the user begin this process with using a technique not well-suited to them
they may become easily disenchanted with this entire process and thus abandon it without having
given it a fair trial. And the entire rationale for undertaking this process is that it can be done in
such a “quick and easy manner” that it will become almost second-nature to the user. (To that end,
some additional types of magnifiers, not mentioned in the text above, are included in the table
below.)
So, let’s review the viewing alternatives so that the user may make a more informed choice:

Method Advantages Disadvantages

Direct Viewing Absolutely Free, easy-to-do.
Phase Telescope May be expensive.
Requires adjustment.
May provide magnification.
Bertrand Lens Free -- if already there!
Very convenient.
Very easy-to-use.
Provides excellent view.
Hand Magnifier Low-cost.
(typ. 10x - 14x) Readily available.
Easy to learn to use.
Basic objective. Commonly available.
(10x, low-cost) Use as Hand Magnifier.
(with Tip toward eyepiece)
Abbe condenser. Often readily available.
(Any old one...) Use as Hand Magnifier.
(with Tip toward eyepiece)
Provides approx. 20x !
(provides bigger image)
Basic eyepiece. Typically readily available.
(10x to 15x/16x) (scope may have extras.)
[See “NOTE:”] Use as Hand Magnifier.
(use ” eyelens-to-eyelens”)
Removing/replacing the eyepiece.
May require switching eyeglasses.
Requires good “ close-up” vision.
Removing/replacing the eyepiece.
May not fit eyetube well (tight).
Viewing may be uncomfortable
(especially for a “ long” telescope).
Very costly to add this feature.
Must be designed for exact scope.
Not available for low-cost scopes.
Requires some skill/practice to use.
Only limited “real” magnification
(depends upon magnifier used).
Requires some skill/practice to use.
Highly-corrected types not suitable.
Basically, equal to Hand Magnifier.
Requires some skill/practice to use.
Not achromatic, small view area.
Iris can become a nuisance,
(must use with iris fully open!)
Only inexpensive types suitable
(Only Non-compensating types).

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 NOTE: The use of an extra eyepiece as a substitute for a hand magnifier is most practical
with the lower-cost microscopes. Often extra eyepieces (15x or 16x) are included with the
scope when new. Any such eyepieces are at least worth a quick “test” so see how they may
perform. In this case, the extra eyepiece is held with its “eyelens” facing the eyepiece on
the microscope. Viewing is then done through the open end of the barrel. However, if such
eyepieces are not available, there is one more option. As the “normal” eyepieces on these
scopes (which typically have binocular body tubes) usually slide in and out very easily,
it is possible to simply pull out the eyepiece from one side and use it as a magnifier for the
eyepiece on the opposite side -- it is then easily returned to the microscope for normal
viewing! Yet, despite this odd use, good images can often be obtained – and for free!

Now, the final selection of the most suitable (or most desirable) method for any user is left to them.
However, in contrasting those methods which require removing the eyepiece vs. those which do
not, it should be pointed out that the eyepieces often fit quite snugly on some microscopes, such
that sliding them in and out can be a bit if a struggle. The same may be said of the fit between some
Phase Telescopes and some microscope tubes. So, for these situations one of the “non-removal”
methods (e.g., Hand Magnifier, or similar) would likely prove most suitable and desirable.

Once the user’s preference has been decided, and practiced a bit, we can then begin to work on
developing an understanding of the indications provided by the technique. And, while the number
of different specific defects made visible is likely to be considerable, these will all fall into
a very few distinct categories, which may be briefly summarized as follows:

Viewed Indication* Probable Cause(s) Remedies

Rear Aperture not filled. Condenser too low or iris closed. Raise Condenser
and/or Adjust Iris.
Iris image not centered. Condenser not centered. Center Condenser.
Objective off-center. Check nosepiece.
Hot Spot in Rear Aperture Illum. light not focused. Check bulb & socket.
If Built-In Illuminator: (Wrong bulb or bulb
not fully into socket).
Can’t fill Rear Aperture Condenser focus wrong. Adjust Condenser.
Wrong or missing Aux Lens. Check Auxiliary Lens.
Iris looks “funny” Condenser focus wrong. Adjust Condenser.
“Glare” inside objective. High-NA objective. Adjust Condenser
and/or Field Iris.

* NOTE: Indications may be affected by specimens. If the “fault” shifts as the

slide is moved, then the specimen is interfering, Move to a clear spot on slide.

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Obviously, this table could be extended considerably and still not cover every possible scenario.
Also, it is not infrequent that there will be multiple problems occurring simultaneously, and no
finite table could hope to cover all the possible variations in such cases. So, what is most important
is that the user become familiar with the normal (proper) appearance of the objective apertures on
their instrument so as to be able to detect when things are not as they should be.
More often than not, the major problems will be found either with low-power objectives, where
uneven illumination often occurs, or (most often) with high-NA objectives, where there is an
overall unsatisfactory image-quality. Naturally, this method cannot reveal every possible problem
which may contribute to a poor image, especially since it is primarily intended to reveal problems
with the illumination.
However, there are some additional common problems which can often result in indications
detectable by this method. These may be briefly summarized as follows:

Viewed Indication Probable Cause(s) Remedies

Tiny circles in Rear Aperture Air bubble(s) in Immersion Oil. Remove, replace oil.
Iris looks closed even when Objective’s internal Iris closed. Open Objective Iris.
Condenser Iris is opened. (only for Iris-equipped objective).
Uneven Darkfield illumination Darkfield condenser off-center. Re-center condenser.
(for Darkfield Condensers). Air bubbles in Condenser oil. Remove, replace oil.
Uneven Darkfield illumination “Patch Stop” not fully inserted. Check “Patch Stop”.
(for Abbe with “Patch Stop”).

Even more uses can be found for this technique, depending upon the user’s skill level, as well as
the particular “viewer” magnification available. Some of these additional uses are:

Verifying Illumination: If everything looks as it should in the rear aperture, then the
problem, whatever it may be, most likely lies elsewhere. Then it’s time to consider other
areas, such as a dirty/defective objective front lens (i.e., grease, fingerprint, damage, etc.).
Phase Ring Alignment: In the absence of a Phase Telescope, other aperture viewing
techniques mentioned in this section may be used instead.
Darkfield Stop Testing: If a user chooses to make his own “ Patch Stop” these methods
may be used to determine both the adequacy of the stop (view light “ leakage”) as well as
reveal any problems with the centering of the stop and/or condenser. Similarly, these
methods may be used for evaluating Rheinberg filters.
Illuminator Lamp Alignment: For scope using illuminators featuring lamp adjustments,
these methods may be used to verify correct adjustment, such as focus and centering.
Condenser Centering: The objective rear aperture view is the way to gauge centering.

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3.10 Illumination and the “Real World”
Here, we talk about illumination again, but now from a “Real World” perspective.
Back in the early 1890s -- Ernst Abbe (of Zeiss) came along and showed (in essence) that it was
only necessary to completely fill the back lens (“Rear Aperture”) of the objective evenly with light
in order to obtain the maximum resolution of the objective.
Naturally, this begs the question, “And just how do we do this ‘back lens’ thing . . . ?”
So, here we go with a bit more of this “Real World” stuff . . .
As Abbe proved so long ago, all you need is a lens system that gives you a NA at least as high as
that of the objective, and which also is just sufficiently well-corrected to fill the rear lens of that
objective evenly with light. Thus the Abbe “Illuminator” (the original name for Abbe’s two-lens
condenser design) came into being (and wide-spread use). Not that this was perfect, mind you, but
it was adequate, and it was cheap to make -- so it appeared as “standard equipment” on a great
many microscopes and thus came into common use.
Now, all this history aside, we can now take a fresh look at the “illumination” problem from a
“Real World” perspective and see more clearly just what’s really going on.
Everyone (in microscopy) seems to think that “ Kohler” illumination is just wonderful, is the “only
way to go,” etc., etc., etc. Nearly every microscopy book printed in the past hundred years has
some fancy illustration of just what “Kohler” illumination is all about, and how they claim it
works.
Naturally, having not invested in such fancy and misleading graphics, I can say pretty much
whatever I wish -- so here’s the “truth” . . . (or, at least, my presentation of it for this book . . .)
First, if all you have is a simple Abbe condenser, you’re somewhat limited, no matter what else you
do. Pouring your money into fancy, high-NA objectives is really not going to make a huge
difference! But, if you actually understand what’s going on, you can achieve truly excellent results
even with just standard stuff . . .
Next, if your scope lacks a costly “Field Iris” (etc.) it may not make all that much difference. It’s
entirely possible that you simply don’t need one -- never mind what all those old textbooks
say! (Modern optics benefit little, if at all, from the use of a Field Iris.)
Again, here’s what’s actually going on (in the “Real World”) . . .
Despite all that you may have previously read about microscope illumination, in the “Real World”
what matters is not where the light comes from but where it ends up . . .
You can get a really great image just using a $3 light bulb for a light source, if you really
understand what your doing! So, it’s not the source that you really need to worry about. You need
to understand what happens after the light gets to the microscope. And, to do that, we need to start
at the place where other books fear to tread -- at the “end.”

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Now, this “end” point is not the eye (or camera), or even the eyepiece -- that is merely a magnifier
that allows us to see this “end point” a bit easier. No, the “end point” is the “intermediate image”
within the microscope. All you can or ever will see of the object is located there. And that is what
makes this point so important. It is the place within the microscope where everything actually
“comes together.”
So, what do we actually find at this point . . . ?
We find the “circle of illumination” produced by the objective. And within this “circle” we have
the magnified image of the object, the result of our illumination methods, and any and all “extra”
stuff (glare, unwanted reflections, artifacts, etc.), as well. (If you aren’t careful in your setup, you
can collect a lot of “garbage” at this point . . . ).
Despite the odd term, “circle” what we have here is rarely sharply defined. Instead, if we could
cut the scope in half, we would find a rather large-diameter image of high-quality (at least over the
area covered by the eyepiece) which gradually degrades and fades away over a total space of 2-3
inches. Thus, this “image” can be much larger than the eyepiece diameter.
Understanding this, we can now see the value of having a “Field Iris” that is focused on the object.
When the image of the object appears at the “intermediate image” point, then the image of the
Field Iris is right there with it. By varying the opening of the Field Iris, it is then possible to “cut
off” any unwanted portions of this image.
“Why do we care about this . . .,” you might ask?
Because the light that produces the “unwanted” portion of the image can easily go astray and cause
all sorts of problems within the microscope which can affect the quality of the image we really
want to see -- glare, reflections, camera artifacts, etc. So, if we have a Field Iris, and use it
properly, we can exercise control over these undesirable factors.
On the other hand, if our instrument (and setup) are such that none of these factors exist, then the
Field Iris becomes completely unnecessary (for that occasion). This means, that microscopes which
lack a Field Iris will perform optimally only under those conditions (optics, specimen, setup) where
no such interferences occur. (One more reason why caution is needed when “ upgrading” any
of these scopes -- high-performance optics are more demanding and can be more
problematic.)
Now, in a transmitted light microscope, there are actually three optical paths to worry about -- not
just the two so frequently depicted in those oversimplified “Kohler” illumination diagrams. Two
of these paths are the ones “everyone” knows about (because that’s all the other books mention).
However, it is the third path that actually matters -- and we have just discussed what lies at the
“end” of this third path.

So now, let’s go on to examine the rest of this new path, as well as review the others . . .

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3.11 Battle of the Irises
There seems to be much general confusion over both the true roles and actual necessities (and uses)
of the two “Irises” commonly found on better (“Kohler illumination”) microscopes. .
The “Condenser Iris” is indispensable for Brightfield microscopy, where it serves as a basic means
for optimizing the image -- balancing contrast, depth-of-field and resolution. But, with other forms
of illumination it may be either non-essential (e.g: Darkfield) or undesirable (phase contrast).
The second Iris, however, a “Field Iris”, is actually a part of the microscope Illuminator. This Iris
serves a very different purpose -- to control glare within the microscope body. It influences the
image’s appearance indirectly, and only in cases where harmful glare may be present without it.
When the Condenser Iris is properly used, its image appears well-defined in the Objective’s Rear
Focal Plane (or, “Rear Aperture”). This is achieved by focusing the Condenser as needed and is
essential to ensure that this Iris can effectively control the Objective’s Working Aperture ( “NA”).
On the other hand, the principles of Kohler Illumination dictate that the Field Iris should be
focused in the Object Plane (the plane of the specimen), so as to control unwanted image “glare.”
Thus, there are two independent tasks that need to be accomplished with the exact same adjustment
-- the focus setting of the microscope Condenser! And thus, the “Battle of the Irises” begins...
In the “Real World” (as opposed to all those “neat-looking” textbook drawings), accomplishing
these two separate goals with the same adjustment of the Condenser is not always easy and, in
some cases, may be nearly impossible!

The fact is, the more crucial these adjustments are, the more difficult it is to
achieve both of them with the same Condenser focus setting -- especially on
a “modern” Lab microscope with a “built-in” illuminator.

Further, as the “working NA” of the Objective and Condenser increase, the relative importance of
the Condenser Iris increases (both in terms of focus, and centering). So, let’s consider, for the
moment, just these two adjustments.
Now, for Kohler Illumination, only in those setups in which the illuminator is separate from the
microscope are the Field Iris and Condenser Iris adjustments truly independent. When the
Illuminator is part of the microscope, most makers only allow one or the other to be centered, and
only very rarely both. (For those scopes where there is no Field Iris, there is only the
Condenser to worry about.)

Unfortunately, many users have been misled to believe that the adjustment and
centering of the Field Iris is somehow essential to obtaining the best images.
Again, only rarely is this the case, and ONLY when such adjustments do not
degrade the Condenser settings, which are nearly always far more important!

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In the setup procedure for a typical modern Lab microscope, the diligent user will generally focus
the image of the Field Iris on the specimen (normally using the 10x objective), and then center the
image of this Iris in the field-of-view. This normally has two effects: (1) it results in the Condenser
often being set too low (limiting its maximum usable NA), and, (2) it usually results in the
Condenser being moved off-center (relative to the Objective) -- both exactly the opposite of what
should be achieved!
Note that for optimum image control (and, therefore, optimal image quality) the proper functioning
of the Condenser Iris should take precedence over that of the Field Iris. This is because the
Condenser Iris directly influences the effective NA of the objective while the Field Iris will
typically affect only some control over image glare (rarely a real problem with modern optics).
However, if the Condenser focus is mis-adjusted, as may occur when attempting to focus the Field
Iris into the Object plane, then the functions of both irises may be impaired. Even worse, it is
possible for the Field Iris to begin to limit the objective NA, a problem that mainly occurs with the
higher-NA objectives, (e.g: 40x/o.65, or more).

Fortunately, this problem is easy to test for – simply adjust the Field Iris while
inspecting the rear aperture of the objective. If the illuminated aperture only
changes in brightness, then the problem is not present. But, if the illuminated
aperture changes in size, then the Field Iris is affecting the objective NA.

Now, in general use, while the Condenser Iris is normally adjusted so as to lie just slightly within
the Objective’s rear aperture, the adjustment of the Field Iris is such that it is normally set just
slightly outside the limits of the visual field. Note that while the Condenser Iris functions by
controlling (limiting) the objective’s Effective NA (or, “aperture”), although it may also
secondarily affect “glare” arising within some older-design objectives.
The Field Iris, however, functions quite differently . . .
Since it is focused in the specimen (or, Object) plane, it defines the outer (visual) limits of the
visual Object Field. Without this “field limiting”, the objective is free to form as large an image
of the object field as it can -- usually much larger than that shown by the eyepiece(s). This results
in an excessively broad “cone of illumination” flooding the interior of the microscope, as well as
the objective itself.
In older microscopes there were often many opportunities within the microscope body for such
excess light to result in unwanted reflections, and thus in “glare,” which reduced image contrast
and quality.
The real function of the Field Iris is simply to remove most of this excess light.
When the Field Iris is properly adjusted, then size of the Intermediate Image is correspondingly
limited, typically to just slightly more than the microscope eyepiece(s) will accept. When this is
done, there is very little uncontrolled light remaining and troublesome“glare” is reduced to
a minimum. (This was Kohler’s reason for adding the Field Iris as part of his “Method of
Illumination”.)

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Now, in Kohler’s Day, this made a great deal of sense and was nearly essential. However, with
most modern microscopes and modern (post-WWII) objectives, this “glare” problem is already
well-controlled by the design of the microscope and the objectives, and so the Field Iris is now far
less important.

Except in critical Research-type applications and/or when certain older, rather

“fussy” objectives are used, a Field Iris will usually make very little difference in the
actual appearance of the final image. – assuming it is not interfering with NA.

At this point it should be noted that errors in Condenser adjustment are not always purely the fault
of the user. In many cases, particularly when a standard “Abbe: type condenser is used, constant
checking and re-focusing of the Condenser may be essential to achieving optimum results.
This may be due to several additional factors (depending upon the exact design of the microscope):
! The Abbe condenser is known for serious “spherical aberrations.” These typically
require compensation (by re-focusing) as different objectives are used.
! The focus of the Field Iris is often affected by simple optics within the Illuminator.
These may result in additional focusing errors which vary with the Iris opening.
! The Condenser may not be exactly (or, fully) corrected for focusing the Field Iris.
Low-cost condensers often have loose tolerances which can affect proper focus.
! Even with a top-quality Condenser, other factors can cause focus9ng errors.
Focus can often be affected by slide, coverglass and mountant thickness.
! For some microscopes, an incorrect “Auxiliary Lens” can lead to focusing errors.
Use of the wrong lens (or none) when one is required) alters Condenser focus.

It should be recalled that the Condenser actually provides three different functions, and that these
may or may not be possible simultaneously:
(1) The Condenser must focus fill the objective Rear Aperture evenly with light.
(2) The Condenser should fill the Object Plane evenly (and fully) with light.
(3) The Condenser should also focus the Field Iris in the Object plane.

In the first case, the Condenser projects an image of its Front Focal Plane (usually, just in front of
its Iris). Here, the Condenser Iris is used to define the size of the Aperture opening and,
consequently, the “working NA” of the objective. This function is critical to proper and effective
use of the Objective.
But, in the second case, the Condenser also attempts to focus the “Field Iris” in the Object
(Specimen) Plane – a very different situation optically. In this mode, purely for purpose of
establishing Kohler illumination, the Condenser attempts to image a distant object (the Field Iris)
at its own Focal Plane. In this mode, the goals are to both fill the Object Field evenly with light and
to also focus the Field Iris in the Object plane – optically a very challenging task!

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Despite the neatness of all the textbook drawings, these two functions can occur at the same
Condenser focus only under very limited conditions – conditions which are rarely fully-known to
the user (e.g: when did you last see a Condenser marked for “Slide Thickness”?).
Now, as if this wasn’t already complicated enough, a further requirement (with or without Kohler)
is for the Condenser Iris to be well-defined in the Objective Rear Aperture. This adds two new
conditions to add to the previous list:
(4) The image of the Condenser Iris should be centered in the Objective Rear Aperture.
(5) The edges of the Iris opening in this image should be (ideally) very well-defined.

However, we can now list these various functions roughly in order of priority:
(1) The Objective Aperture should be filled evenly with illumination.
This allows maximum use of the available Objective NA.
(2) The Condenser Iris should be clearly imaged in the Objective Aperture.
This allows the Condenser Iris to properly control the Objective NA.
(3) The Condenser Iris should be centered in the Objective Aperture.
This allows the Condenser Iris to properly control NA and image contrast.
(4) The Condenser should fill the Object Plane evenly with illumination.
This may prove difficult with low-power objectives on low-cost scopes.
(5) The image of the Field Iris should be centered and focused in the Object Plane.
This may be difficult to do with an Abbe condenser and is least important.

Here, the first two requirements here are purely a matter of Condenser Focus adjustment.
(For further details on this, refer to Section 3.8, “Verifying Your Illumination”.)
The third item, Condenser centering, may not be possible -- many modern Lab microscopes lack
any provision for user adjustments of this kind. However, if such adjustments are provided, note
that this function should take priority over the easier and more common centering of the Field Iris.
Furthermore, the lack of color corrections in the standard Abbe condenser may preclude
accurate focusing of the Field Iris, since the image of the Field Iris edges may change colors
dramatically, making the determination of true “focus” nearly impossible, especially with
higher-NA objectives!
Evenly illuminating the Object space (fourth item) is generally not a problem except with low-
power objectives (e.g: 4x or less). This relates to the size of the image of the Illuminator light port
formed in the Object plane. Occasionally, this image is not quite large enough to fill the entire field
of view – particularly if the scope has been fitted with new eyepieces have a wider visual field than
the original type. (In such cases, one easy “fix” is to fit a simple diffuser over the lower end of
the condenser to expand the effective size of the light source.)

Because of all these difficulties, and all the factors involved, the best choice, if optimal images are
desired, is simply to routinely inspect the Objective Rear Aperture to verify that all the relevant
adjustments have been properly made! (See Secs. 3.8 & 3.9 for appropriate methods.)

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3.12 Kohler and Glare
Starting with the simple Abbe condenser, we can analyze its “Real World” operation. To do this,
we simply begin at the most critical point in the microscope, the objective’s “Rear Aperture” (or,
“Rear Focal Plane”), and work our way backwards toward the light source.
If we have a moderate-aperture objective, say NA 0.65 or less, we find that the aberrations of the
usual Abbe Condenser are not terribly significant, and this inexpensive design will function nearly
as well (for general use) as more costly condensers. This is because the Abbe, up to NA 0.65-0.70,
can easily accomplish the critical goal of “uniformly filling” the objective’s rear aperture with
illumination. (Note very carefully the word, “uniformly” -- that is very important.)
With the objective’s rear aperture fully illuminated (as above) the remaining problems are the
adjustment of the illumination to minimize any “glare” (excess, scattered background light in the
final image) as well as to control the “Effective NA” of the objective. Normally, these goals are
independent. But, if the NA is reduced noticeably, this also tends to reduce associated glare
in the image, as well as boost the overall contrast level.

Kohler chose to separate these two function by using a “Field Iris” in addition to the
standard “Condenser Iris.” The reason for the “Field Iris” is to provide an added
measure of “glare control” beyond that possible with just the Condenser iris.

As discussed earlier, the Abbe condenser’s Iris may or may not be effective (depending largely
upon condenser’s focusing) due to the Abbe’s inherent “spherical aberration.” The higher the NA,
the more significant this problem becomes. Also, this same “spherical aberration” problem may
also result in reduced control over “glare” as well .
Fortunately, it is relatively easy to check your instrument for its sensitivity to such “glare.”
All that is required to perform this test is simply to configure your instrument properly for
observations and then to open up the Field Iris (if your scope has one) while observing the object.
If the image contrast declines significantly, then you may have a “glare” problem, and this is best
managed by correct use of the Field Iris. (See Sec. 3.8 for effective methods to observe such
“glare”.)
However, if the image contrast barely changes (or you have no Field Iris and the image still looks
just fine) then the Field Iris is not performing any useful function and need not be used – simply
leave it opened. (This latter case also neatly solves the problem of false colors in the object field
caused by the condenser’s poor imaging of the Field Iris -- but, remember that this situation can
change with both the object viewed and the objective used . . . )
Modern microscopes (equipped with modern optics) rarely benefit from elaborate illumination
methods to the same extent as they once did. This is also true for today’s well-engineered
“standard” microscopes having simplified illuminators. (In today’s competitive world, if you don’t
have a good image you just can’t sell your instruments -- so, competitive pressures have been a
powerful force in addressing the classic “glare” and image contrast issues.) Just be aware that this
does not mean that one can apply high-end (or, “Research-grade”) objectives on a “routine” Lab

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microscope with impunity -- some of those optics require very careful adjustment of illumination
to minimize glare [no names mentioned] . . .
Very simply that the “glare control” benefits of Kohler’s illumination method are much less
relevant than they once were. And, if there’s no “glare” problem, then there’ really no need to have
a “Field Iris” -- unless you plan someday to use a lot of older objectives which just may have such
problems! In such cases the Field Iris can be very effective -- but only if and when it actually is
needed!
Now, consider the modern clinical laboratory (“Lab”) scope, for which convenience and ease-of-
use (as well as modest cost) are considered paramount. For these reasons, plus the fact that “true”
Kohler illumination no longer offers the obvious benefits that it once did (for most lab specimens),
that makers rarely include such “real” Kohler capabilities in their scopes these days. Instead, what
they provide may be considered as simply an “approximation” of Kohler illumination – typically
with some level of non-removable diffusion inserted somewhere in the Illuminator optics.
When Kohler illumination is set up in the classical (or, “true”) way, a sharp image of the lamp
filaments appears in the rear of the objective. This, however, requires not only a well-corrected
system of lenses within the illuminator (a “lamp condenser”) but also the addition of adjustments
to allow proper setup and control.
While this approach had some real merits back in Kohler’s day (late 1800's to early 1900's) there
are a number of today’s methods for which this exact method is entirely unsuitable. (These include
phase contrast, DIC, Darkfield, HMC, etc.) The problem today with this “true” form of Kohler is
that the presence of such visible “structure” ( image of the lamp filaments) in the front of the
Condenser violates the basic requirements for uniform illumination of the Condenser aperture with
these modern methods. (This is because any such source “structure” can give rise to unwanted
artifacts in the final image.)
Now, adding a diffuser in the illumination path not only removes eliminates the need for
adjustments, but also removes any lamp filament “structure” from the microscope’s illumination
and image paths. Thus, many of today’s microscopes incorporate some form of diffused
illumination, simplifying their use, broadening their application, and also reducing their cost.

Inasmuch as “true” Kohler illumination is likely to be needed almost solely in

Research applications, the implementation of this capability is now largely limited to
the most costly Research-grade instruments, and only a few of those!

Kohler illumination can perform superbly when used for objects like bacteria, blood films,
diatoms, etc., which tend to occupy only a small portion of the total visual field area, and thus
leave much open area for the undiffused background light pass. However, for those objects which
cover much, or most, of the visual area, and thus provides little or no “empty” space for the
background light, the beam then becomes mostly “diffused” by the specimen material, and thus the
potential advantages of Kohler lighting are largely nullified.

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Notes

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Notes

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4. “Real World” Microscope Use
For readers who may have jumped to this chapter without fully reading the preceding
Chapters, selected parts of that material is re-discussed here for their benefit.

4.1 The Compound Microscope

A very commonly-held misconception is that microscopes are employed in an effort to “make
things bigger.” While this may be true to some extent, it is also largely a secondary consideration.
The “real” purpose behind a microscope is to allow the user to see finer detail.

NOTE: In microscopy, the ability to discern fine detail is termed, “resolving power,” or
“resolution.” This is quite different from “magnification,” or, simply making things look
bigger. (There are a few technical terms in microscopy which you really should know. For
some help understanding these terms, see Appendix I -- Glossary.)

Now, one of the main features of the modern compound microscope is its ability to provide very
detailed images. However, it can only accomplish this when it is operated under the proper
conditions. Also, the greater the level of detail expected, the more carefully it must be used. If not
used properly, image quality can suffer significantly, so an understanding of the correct use is
important if one is to achieve the best results.
There are two basic subgroups for compound microscopes -- a “Biological” (or, “transmitted
light”) form and an industrial (or, “reflected light”) form. Naturally, these differ in whether the
object is transparent with illumination through the object, or opaque, with illumination reflected
from the object. The biological form is the more common, and the one most often used by
amateurs, so we shall concentrate solely on that type in the following discussion.
Today, almost all Biological microscopes include some form of “built-in” illumination. This is
usually (but not always) self-contained within the base of the instrument. Also there is often some
form control over the illumination intensity. These are useful features as they simplify both the
setup and use of the instrument -- and also tend to limit the number of mistakes than can be made!
Assuming the instrument is in proper mechanical and optical condition, and the specimen to be
observed has been properly prepared, then the most critical factor governing the quality of the final
image will be the illumination (as discussed earlier). Basically, this involves the Illuminator and
the Condenser, and these will be discussed shortly. Just remember, if you don’t get it exactly right
at first, you can probably still get decent results. But if it isn’t somewhere near right, you will
likely not get anywhere near the image quality you should.
Thus, in most respects, the level of performance of the microscope is dependent upon the care (and
skill) with which it is set up. The purpose of this chapter is to review the various steps required to
achieve proper set up while imparting some understanding as to why these are necessary. (Limited
experience is assumed.) The assumption is also made that the user has a “modern” instrument with
a “built-in” illuminator, but other than that, most of the procedures will be valid for other types of
instruments as well. So, let’s begin at the bottom (with the illuminator) and work our way up...

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4.2 Basic Illumination Setup
We begin with the scope’s built-in illuminator. Here, there are many forms, which may differ
significantly in their details, but which all end up with a light that shines up into the main part of
the instrument. Some of the more elaborate stands will have a “Field Iris” at or near the top of the
illuminator. This controls the visible diameter of the light beam exiting the illuminator. For now,
this should be opened to near maximum. (If your scope lacks a Field Iris, don’t worry -- most
scopes do not have one. In this case, simply ignore any comments relating to the use of the
Field Iris and its use. For more information on the Field Iris, see Section 3.10.)
Above the illuminator port is a lens system called a “Condenser.” On the lowest-cost stands this
may be just a simple lens or two mounted directly on the underside of the stage (the “table” that
holds the specimen). Compound microscopes which lack a condenser are called “toys” (unless they
are stereo microscopes). The purpose of the condenser is to control the illumination of the
specimen (object) so that maximum detail may be seen. If the condenser is absent, or improperly
used, then image quality will suffer, or at least be quite limited.
Initially the condenser should be focused just below its uppermost position (closest to the
specimen). This is always a “safe” starting point, and usually a reasonably accurate placement as
well. Condenser focus is usually controlled by a focus knob on one side of the mechanical
mounting which holds it. (On low-cost scopes it the condenser focus may not be adjustable, or may
be adjusted over a limited range by carefully rotating the condenser in its mount.)
One additional Condenser control is the “Iris.” This is a variable opening, usually located near the
bottom of the condenser and controlled by a small lever (or by rotating a knurled ring) on the
condenser body. Initially, this should be set such that the iris is at near-maximum opening. (Many
users believe that this is used to control the illumination intensity, which is incorrect. Basically,
it is used to control image contrast and minimize glare -- but more on that later . . . )

4.3 The Ubiquitous Abbe

There must be, by now, “zillions” of Abbe condensers in the world. Developed late in the 19th
century by Ernst Abbe (of Zeiss), it was then termed the “Abbe illuminator.” It is a “cheap” (low
cost) optic -- just two easy-to-manufacture lenses and a simple iris, but it has survived for well over
100 years, basically unchanged. So it must have a lot going for it, right . . . ? Wrong!!!
It’s cheap to make and easy to use. And back in the days when most microscopes featured very
large mirrors that would swing to all sorts of angles, it was a big improvement. But that was 100
years ago. From an optical standpoint, its really quite limited. It is an “uncorrected” system. It has
huge aberrations (optical errors). If you don’t believe me -- then prove it to yourself . . .
Take your condenser (carefully) from its holder and try to use it as a magnifier. Open the iris and
hold the large end to your eye while examining a newspaper or magazine photo (with lots of tiny,
regular dots). Notice anything funny? Move the condenser up and down a bit, slowly, to alter the
focus. See how the image of the dots changes, how the focus shifts? With the Abbe condenser, the
focus is different for different parts of its field, changing radially away from the center (optical
axis). Thus, there is no position at which the entire field of the Abbe is in focus at one time.

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Now, reverse the condenser and look through it at a light source (holding the small end toward you
eye). As you do this, open and close the iris. With a good-quality Abbe, the iris should be fairly
distinct as it becomes nearly closed. Ideally, when the eye, condenser and light course are all on
the same axis, the closed iris will appear rather sharply defined (but not perfect) and will be
surrounded by an almost all-black area (the closed iris). A cheap Abbe can have an unbelievable
amount of stray light under these conditions -- in that case, no black, just whitish-gray.
The next step is to open the iris slowly and observe the quality of the image of the iris edge. Notice
how the image gets worse. The larger the iris opening the worse the image. And, as the iris is
opened past about 2/3, the image begins to get really bad. Past about 3/4 there’s so much glare that
it’s very hard to be sure just where the iris opening actually is.
At this stage you’re probably reluctant to put that “terrible” Abbe back on your scope. but you
should do so anyway. The purpose of this exercise was to demonstrate that the Abbe condenser has
some very serious limitations, but you should also be aware that when it is used within these
limitations it can work very well. The net result of all this is that the Abbe works quite well up
to a limit of about NA 0.65 and beyond that its performance is increasingly handicapped by
its inherent aberrations.
In the microscope, what all this means is that there’s simply no way you can utilize the full rated
NA of any objective higher than about NA 0.65 or 0.70 if you’re using an Abbe condenser. This
is because the critical optical conditions necessary simply cannot be created when using the Abbe.
(Although there are better condensers made, which far surpass the Abbe in performance, they also
tend to be quite costly and are made for only a few higher-grade microscopes. Fortunately, if you
learn how to use the Abbe properly, you will probably never need to buy one!)
The secret to using the Abbe (or any condenser, for that matter) is very simple: the condenser must
fill the “Rear Aperture” of the objective evenly and fully (or nearly so) with light. This condition
is necessary if you want to achieve the maximum NA from the objective. If you cannot achieve
this, then the NA will be something less than that marked on the objective, and sometimes much,
much less. (See Sections 1.10 and 3.7-3.8 for more on Illumination.)
Now, with the condenser set-up and with everything open, and with the illuminator ON and set for
medium intensity, we are ready to begin looking at something . . .

4.4 The Binocular Body

The first step in looking through a microscope is to verify the eyepiece adjustment. On simpler
(“monocular”) microscopes this is easy -- there’s just nothing to do! However, most microscopes
these days are equipped with “binocular” body tubes. Unfortunately, for many users they are
simply more trouble than they are worth . . .
While these hold two eyepieces, this has nothing to do with “stereo” vision -- these binocular
bodies are primarily there because buyers expect to have them and makers like to sell them ($)!

NOTE: There is no “stereo” because “stereoscopic vision” requires two different image
paths. In a biological microscope, both eyepieces share the same image, thus no stereo!

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The binocular body was developed based on the somewhat misguided notion that “binocular”
vision (as opposed to true “stereo” vision) was always superior to “monocular” vision (viewing
with just one eye), and that this was therefore the ideal eyepiece configuration for all users.
Now, “binocular vision” entails viewing the same image with both eyes, while true “stereo vision”
entails having a slightly different view of the subject for each eye. It is the difference in these two
views that accounts for “depth perception” and allows distance estimation -- abilities essential to
the survival of a hunting animal. And since conventional microscopes can provide only a single,
common view of the object, no matter how may eyepieces they feature (some teaching scopes can
have 10 or more!), there can be no true “stereo” image from which to benefit. (For a “true”
Stereo image you must use a real “Stereo Microscope,” which has two separate optical paths.)
The real benefit of a binocular body is that it may lessen eyestrain somewhat for those who use the
instrument for extended periods, assuming that (a) the binocular setup is correctly adjusted, and
(b) that they are able to adapt their vision to the typical, parallel binocular eye tubes!
There is also another factor here which few bother to consider -- a high percentage of the
population has a “dominant” eye. For such users, one eye is “preferred” by the brain and that one
is the one used primarily for the collection of image detail. This is normally the user’s “stronger”
eye; the eye which has the best vision. For these users, the second eyepiece on the binocular body
is of little benefit and can easily become a liability (if incorrectly adjusted).
The most common result of any of these problems is “eyestrain” And, although some users seem
to be able to train their eyes to adapt to the situation, this does not alter the fact that for many users
the binocular body actually provides no real benefit.
Although it is usually possible to adjust the binocular setup to accommodate the individual user,
many users have a limited understanding of how to do this. Worse, if their eyes are unable to adapt
to the somewhat “abnormal” (“ parallel”) eye tube arrangement, then no amount of adjustment can
result in a satisfactory result. (The situation can become even more complicated if the user wears
eyeglasses.)

NOTE: Because of this “convergence” problem, where some user’s eyes simply
cannot adapt to parallel eyetubes, some makers (AO/Spencer, for one) once
provided binocular bodies with “converging” eyetubes, which allowed the users
eyes to function at a more natural angle when viewing through the microscope.
However, these are now mainly items of nostalgia.

Now, there are also users who simply cannot acclimate themselves to using a “monocular” (single
eyepiece) scope. These users typically do not have a “dominant eye” and may have either very
good vision in both eyes, or similar vision deficits on both eyes. In either case, the brain shows
little preference for one eye over the other and thus finds it difficult to adapt to an artificial image
which is presented to just one of their eyes.

The end result of all this is that there is simply no single solution which works equally well for
all users -- and so it is up to the individual user to determine what works best for them.

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4.5 Binocular Adjustment
The correct adjustment of a binocular microscope body requires balancing a number of factors
such that the end result is optimized for the individual user. However, this balance may be (and
usually is) somewhat different for each individual -- hence, the need to know just how to
accomplish this.
The first step is adjusting the “interpupillary distance.” This sets the distance between the
eyepieces approximately equal to the distance between the user’s eyes. When this is accomplished,
the views from the two separate eyepieces will “merge” into a single image. (However, some users
are simply unable to achieve this result, for reasons mentioned earlier.)
The next step, unfortunately, requires just a bit of knowledge . . .
There are two types of binocular microscope bodies. Buyers may think of them as “cheap” and
“expensive,” but their formal designations are “Jentzsch” and “Siedentopf” -- however,
there’s no need to remember these fancy names, only the differences between the two.
In the more common type (Jentzsch) the two eyepieces slide sideways for adjustment. This is an
easy mechanism to build, is lower in manufacturing cost and does the job well enough -- so, this
has become the more common type. However, it has one small problem, a consequence of its
simplicity; the distance from the objective to each eyepiece (the “ tube length”) changes as the eye-
piece separation is adjusted. For this reason, these bodies usually also provide adjustment collars
on both eye tubes so that the proper “correction” may be set by the user, after adjusting the
eyepiece spacing.
Once the eyepiece spacing has been set the corresponding distance is read from a small scale
provided on the binocular body, and this number is then transferred to each eyepiece tube by the
user. After this has been accomplished, one of the eye tubes (normally for the non-dominant eye)
may be further adjusted to accommodate the focal differences between the user’s two eyes.
Now, since you might suspect that some buyers might be concerned about performing such a
complicated process, a number of manufacturers (AO, Leitz, Olympus, etc.) designed mechanisms
into their binocular bodies to automatically adjust the “tube length” as the separation is varied.
This allows them to retain the economics of the original design and still simplify its use (at some
additional cost, of course).
Naturally, the alternative body type (Siedentopf) accomplishes this automatically (optically, as a
function of its design, rather than mechanically). This type is also known as the “constant tube
length” type body, for just that reason. It is characterized by a “hinged” appearance where the two
sides of the unit are hinged about a central section. The eyepiece spacing is then adjusted simply
by “folding” (or “unfolding”) the unit. (Manufacturers who offer this type of body are Zeiss,
Nikon, and now Olympus, just as examples.)
As with the other type, once the eyepiece separation has been set it may be necessary to adjust one
of the eye tubes so that both eyes view a focused image simultaneously. Otherwise, there is simply
not much point in having a binocular body!

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However, note that on some bodies (Zeiss, Nikon) the adjustment may be provided via the
eyepieces themselves, rather than on the binocular body. These typically offer an “index” mark (or
line) as a guide, and may or may not have additional markings. With these it is usually good
practice to check both eyepieces (set them to their “zero,” or their “nominal” settings) before
adjusting the binocular spacing, as well as before each use of the instrument. (Just remember that
“swapping” eyepieces of this type “side-to-side” will likely disrupt any prior focus adjustments you
might have made and consequently require a re-adjustment. )
It makes little difference which binocular body type is used -- all accomplish the same result in the
end. The main attraction of the simpler types is that, since the eyetube adjustments are on the
binocular body itself, the more-costly adjustable (“focusing”) eyepieces are not required. Thus, if
it is likely that different eyepieces will be used on a particular instrument, then there would be
some economic advantage to selecting the lower-cost body type. And if the instrument is intended
primarily for exclusive use by one user, then any need for readjustment would be rare and there
would be little advantage to the more costly body type (which is intended for situations where an
instrument is likely to be frequently shared among two or more users, in which case automatic
adjustment could be of some advantage).
So, now that we have skimmed through some of the basics, and adjusted the binocular body
(assuming one) for proper viewing, it’s time to begin putting all the pieces together and begin
actually using the instrument.

4.6 Low-Power Observation (4x objective)

Most modern microscopes are equipped with a low-power (or, ”scanner”) objective of 4x, or so.
This is usually the shortest lens on the microscope “turret,” and is typically marked, “4x/0.10" or
something fairly similar (Zeiss typically uses 2.5x, which is close enough for this exercise).
The “4x” makes a good starting point for several reasons: it is the most forgiving of errors, which
makes it the easiest objective to work with, and it reaches focus a comfortable distance from the
object. Because of this large “working distance” it is easy to view the object under study directly,
as well as through the instrument. This makes it easier to relate object movement and illumination
changes to the eyepiece view. It also has sufficient light-gathering power to allow some level of
image formation based on ambient illumination -- so there is likely to be some sort of image (even
if a bit dim) regardless of how improperly the illumination may be set up initially!
So, as a first step, some sort of test object is needed. For the novice, the object may be as simple
as a small piece of a newspaper or magazine page, mounted onto a glass “slide” using transparent
tape. For more advanced users it can be a prepared slide having any object large enough to be
visible to the naked eye. (The requirements are that the object be large enough so that its
motion can be seen and so that its illumination can also be seen directly.)
The test object is placed on the microscope “stage,” with the object on the side closest to the
objective lens. The instrument is initially arranged as outlined in the previous chapter and the “4x”
objective should be rotated into position above the object.
The following list summarizes the basic requirements (as briefly as possible) . . .

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Starting at the bottom of the microscope and working up, for basic setup we need to have:

Microscope Basic Setup Requirements

(a) Some illumination for the object -- not too much and not too little, for a start.
(b) The condenser is in position to focus the light on the object -- just below the slide.
(c) The desired objective lens (e.g: 4x) is in position over the object.
(d) The binocular eyepieces have been adjusted (at least for spacing).

Even more simply, you just need to get some light properly on the object and you need to get the
eyepieces at least spaced properly for your eyes. And that’s all you have to do to begin . . .
On most modern scopes, “Coarse” focusing is accomplished by moving the object stage up or
down until the object lies at the correct “Working Distance” for the objective in use. You’ll know
when this distance is correct because at that point the object should appear in sharp focus. (For
many modern scopes, object focus occurs when the face of the nosepiece is 45mm above the
object. That’s simply because the new “DIN” standard says that the objectives must work like that.
(For more detail on this, see Section 2.8 -- Parfocal Distance.)
So, with the 4x objective in position over the object by setting the stage such that the face of the
nosepiece (more precisely, the rear of the objective, just where it screws into the nosepiece) is a
bit less than 2-inches above the slide there should be something in view when looking into the
instrument. For some microscopes this distance might be more or less. (If you see nothing, just
try moving the object around slightly on the stage. This is why a fairly large object was
recommended.)
When something moves into view (at this point, most likely blurred) the next step is to adjust the
focus as needed to achieve a sharp image. Focus should be set so that the center of the field of view
is in sharp focus. (Most or all of the field may be in focus, but the center should be sharp.)
Now that there is something there to be viewed, the next step is to check the eyepieces and make
any final adjustments needed there. The binocular body should be set such that the “image circles”
from the two eyepieces merge into a single field-of-view. (Be forewarned that for a few people,
this is just not going to happen . . . In that case, either cover one eyepiece or else remove it
and fit a dust plug into the opening to protect the microscope optics.)
Once there is a proper “field-of-view,” the eye tubes (or eyepieces, if they are adjustable) should
be set so that both eyes see the sharpest image possible. An easy way to test for this is to
alternately cover the eyepieces (first one, then the other) with a piece of paper (a business card
works well) and set the eyepiece adjustments for maximum sharpness, leaving the microscope’s
focus unchanged. You can close one eye and then the other, but for some people this will alter the
eye’s focus slightly, and thus result in a less-than-optimal result. If you should have a “dominant”
(or, “stronger”) eye, always focus the microscope for the sharpest image using that eye.
At this point the instrument is perfectly usable, although the image may be a bit less than perfect.
The next step is to refine the adjustments to obtain a good image. For the 4x objective, there just
isn’t much to adjust, mainly the condenser, and so, here we learn again . . .

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The typical “Abbe-type” condenser has a tough time evenly illuminating the entire field of the 4x
objective. For lower-priced instruments there may always be a certain degree of unevenness in the
illumination when using this objective. This is most usually seen as a slight darkening toward the
edges of the field. However, if the shading is not symmetrical, or there is a pronounced “hot spot,”
then there may be a problem with the scope’s illuminator (lamp off-center, or mis-adjusted -- see
your owner’s manual for proper corrective action).
To some extent this unevenness may be reduced (or perhaps eliminated) by adjusting the
condenser. If the condenser’s “iris” is nearly closed, that may the one problem -- the iris needs to
be opened. However, it is more likely that the condenser’s height (focus) simply needs to be
adjusted -- so simply raise the condenser until it nearly touches the underside of the slide and then
lower it while viewing the microscope image. (A bit if “fine tuning” should result in fairly even
illumination.)
For the typical microscope which utilizes the simple “ground glass” type illuminator, there isn’t
much that can go wrong -- just avoid setting the condenser set such that the rough “ground” surface
(or any dust that may be present) ends up being focused on the object being viewed!
Now, instruments having more complex illumination systems will likely require a more complex
set up procedure, but that lies beyond the scope of this introduction. What should have been
accomplished at this point is to arrive at a basic, correct instrument setup which yields a decent
image with the 4x objective. Having accomplished that, we can move on . . .

4.7 Medium Power Observation (10x objective)

Once the 4x objective is in focus and the remainder of the setup is satisfactory, switching to the
10x objective (by simply rotating it into position) should result in an image that is roughly in focus.
At this point simply adjusting the instrument’s “ Fine focus” should result in a sharp image of the
object. (Note: In some cases, a slight change of the Coarse focus may be needed.)
As with the 4x objective, there isn’t much “fine-tuning” to be done with the 10x objective which
can really optimize the image, except perhaps for adjusting the condenser. And the same comments
which apply to adjusting the condenser for the 4x objective can apply here as well, except that now
we have arrived at the optical threshold where the condenser iris can begin to play its role. Once
the condenser has been adjusted for even illumination (which should be fairly close to the top of
its range of travel), we can then adjust the iris opening to optimize the image.
Most literature expounds on this simple exercise, often accompanied by professional-looking
drawings of what one should see here of there. All of that is well and good, but there is a very easy
and simple way to do this. (Again, refer to Chapter 3 for more details on this.)
In a microscope, not all of the light that enters the objective will result in a useful image.
Especially on instruments with basic illuminator systems, extra light can flood the objective and
result in “glare” which reduces image contrast. Now, the condenser “ iris” can be used to control
this glare and thus achieve a balance between image resolution (the ability to see very fine detail)
and image contrast. And there is just no easier way to do this than simply by viewing the object
through the microscope while adjusting the iris! (See Section 3.8 for more information.)

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First, as a learning exercise, this should be done over the full range of the iris. Note that with the
10x objective the iris has no effect over a large part of its range -- from fully open to about 2/3
closed. (This is because there is only so much light that can enter the 10x objective and once
that point has been reached opening the iris even further has no effect.)
Now, as the iris is closed more completely, it will be noted that the image darkens -- not surprising
since the light is being cutoff by the iris. However, if the iris is opened and closed carefully, it will
be noticed that there is a narrow range where there is a sudden transition between the region where
the iris has no effect (overly “open”) and the region where closing the iris darkens the image.
This “transition” region marks the threshold where the iris allows just enough light to pass to
permit the objective to operate at maximum resolution, and where further closing of the iris results
in higher image contrast but some loss of resolution. This is where the iris should be set -- for the
best image. With a bit of practice this “optimal” setting for the iris can be found almost
instinctively. It lies at the point where the background illumination can first be seen to decrease
(slightly) as the iris is closed.
Closing the iris a bit further will tend to lower the illumination a bit further, but also result in some
increase in overall image contrast. The optimum point depends on the object, the objective, the
illumination and user. No matter where the iris is set, it is a compromise. There is no absolute right
or wrong (despite what some texts would indicate) -- it’s all a matter of the user’s judgement.
Now, to be fair, what the books are warning of is the fact that closing the iris too far will result in
an optical effect termed, “fringing” (“diffraction fringes,” actually) where the edges of object
details are grossly exaggerated. However, depending on the object and the desired results, a bit of
this may not be all that undesirable. (Typically, this mode is employed to view transparent
objects, such as protozoa or diatoms, as an expedient to gaining higher image contrast).
A bit of experimentation with this, using various test objects, should quickly reveal the “optimal”
setting for your iris, and just how this “threshold” appears through your microscope eyepiece(es).
For really critical work one might try removing an eyepiece and peering down into the instrument
to view the rear aperture of the objective as the iris is manipulated, but I would suggest that you
first try to adjust the iris by viewing the object normally, and then you can try the “aperture
viewing” (as some texts suggest) to confirm your results.
Personally, I never could decide whether the iris was (or should be) set at 5/8 or 3/4 or 47/64 of
the objective aperture by viewing without the eyepiece, but I could very easily set the iris nearly
optimally simply by viewing the image normally. After all, the “best” iris setting depends mainly
on the appearance of the object -- which is exactly what all of this is all really about, isn’t it?

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4.8 High-Power Observation (40x objective)
This brings us to the threshold of “serious” microscopy. Her, we are crossing the threshold into the
region where results can depend more on the user’s skill than on the equipment used!
Most people don’t understand (or don’t want to believe) this, but the old, tried-and-true 40x “high
dry” objective (typically, “40x/0.65") will show you about 90% of what you can see with any light
standard microscope!
This, of course, assumes that you really know how to use that objective properly, and how to
respect its limitations. Fortunately, there is not all that much to learn, and it’s not at all difficult
to understand.
Now, with the 40x objective, a number of factors converge to complicate things (just a bit)...
First, this is the most powerful objective that the average microscope can fully exploit. The reason
is that the typical “Abbe-type” condenser reaches some of its optical limits with this lens, as does
the typical “basic” (“ground glass”) illuminator systems commonly found on low-cost scopes.
Second, regardless of the lens employed, there actually has to be “something there to be seen” that
can’t be seen with a lesser lens. Thus, even with the finest optics available, it is only possible to
improve on the results of the 40x objective by a factor of two. And while that may seem like a lot,
what we are really discussing are only those details which lie within that very narrow range.
Finer details simply cannot be seen with an ordinary light microscope, and coarser details are
easily seen using a (40x or even lower-power) objective. Thus, there is a very narrow “range of
exclusion” here – everything below that range can be seen with the 40x objective, or less. (For
anything significantly above that range, you would need an electron microscope . . .)
Third, there is the matter of specimen preparation. Preparation for viewing with a 40x objective
is not particularly demanding, but the necessary preparation for more exotic lenses can easily lie
beyond the means of all but well-funded laboratories. After all, just because something’s there,
doesn’t mean that it’s easy to see, and many of the most challenging structures require difficult
sample preparation processes to make them visible.
Now, none of this is meant to imply that there’s no point to having a “better” objective or even a
“better” scope. The point is that there’s just not much left that requires such equipment in order
to make it visible -- once you learn how to get the most out of your 40x objective!
Also, of the 90% or so of objects that are visible, the average user only gets to see a limited
selection of those, either because of lack of access (to the samples), lack of opportunity, or a lack
of interest. So, while it’s only human nature to want a bigger house, a faster car, or a better
microscope, it might also be prudent to recall that there’s something to be said for learning how
to get the best out of what one already has!
All that said, we can now return to the original subject, which was how to properly use this
“all-around workhorse” (40x) objective . . .

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The comments which apply to the 10x objective apply even more strongly to the 40x. Here, for the
first time, the user’s skill and knowledge and materially effect the quality of the results achieved.
Still, there is also nothing really new here to worry about -- it’s mainly just paying a bit more
attention to detail and taking a bit more care in setup.
Much more so than with the lower power objectives, the quality of results will depend on the
proper use of the condenser -- and especially the condenser “iris.” To understand why, we need to
digress just a bit into some optical theory, but again, painlessly . . .
The ability of a microscope objective to resolve fine detail depends mostly on just how much light
it can capture from the object. Now, here we’re not talking about light intensity (brightness) but
about something called, “aperture.” As an analogy, with a telescope this ability is a function of the
size (diameter) of the main lens (or mirror, depending on the design). The larger the diameter the
finer the details that may be seen.
But in a microscope this “aperture” is buried deep within the optics of the objective. So that users’
can determine the useful aperture, a number (that’s all it really is, just a number), termed,
“numerical aperture” (or, “NA”) is commonly marked on the objective barrel. This allows users
to easily determine the relative “resolving power” (ability to see fine detail) of each objective.
That’s what those “odd” numbers (i.e.: 0.10, 0.25, 0.65. etc.) marked on the objectives next to the
magnification are, and the larger the NA number, the better the “resolving power”. In fact, it is
entirely possible to calculate the smallest detail that can be seen by any particular objective if you
know its NA. Fortunately, all we need to understand is that at NA 0.25 you can see details that are
2.5 times finer than you can with NA 0.10. Also, at NA 0.65 you can see details that are about 2.6
times finer than you can with NA 0.25. In other words, the resolving power tracks the NA’s.
Now, you might have noticed something else here, although it was not explicitly stated. The NA
0.10 is a property of the 10x objective, and the NA 0.65 belongs to the 40x. So, while before you
might have thought that you could see something 4-times small with the 40x (vs. the 10x), the
NA’s tell a different story here, the truth. The 40x objective “resolves” fine details only about 2.6x
“better” than the 10x! (If you want to get a true 4x improvement over the 10x objective, then you
must use a “ 40x/1.00" objective -- which is not exactly inexpensive, and also has its own
considerations.)
At this point we can take one more step back, and then tie all this together.
Recall having seen those pictures of a condenser iris and the “rear aperture” views of objectives
that commonly appear in microscope books (or on the Internet)? Well, this is what they were
alluding to. The resolving power of your objective is limited by its “Numerical Aperture” (“NA”),
and that, in turn, is linked to the condenser. And while the condenser cannot “improve” on the
“NA” of an objective, it sure can reduce its effective value in a hurry if improperly used! The
reason for this lies in the fact that the NA of the condenser is affected by two factors, its focus and
its iris opening. (Again, all of this is covered in more detail in Chapter 3.)
This is because, in a microscope, certain conditions exist where the condenser’s “Effective NA”
can limit the usable NA of the objective. So, just because the NA is marked 0.65 doesn’t guarantee
that 0.65 is what you’re getting. If the condenser is improperly used you may actually
be getting significantly less.

I-83
And this is why operation of the Condenser Iris was stressed earlier. It is a minor factor with the
10x objective, but it becomes much more important with the 40x (and is very important at 100x).
So, now that you can understand the importance of all this, and later (below) we can discuss a few
additional factors that determine the effective NA of the objective when working with the 100x.

4.9 The Oil Immersion Objective (100x objective)

Since I have painstakingly denigrated the ubiquitous Abbe condenser (above) it should come as
now real surprise that I’m not all that enthused about using “oil immersion” on common lab scopes
(unless there’s a genuine need for it). And, as usual, I have several reasons for this.
First, it makes a mess -- there’s oil on the objective (to wipe off later) and oil on the slide (to clean
off ASAP), as well as possibly oil all over the stage and perhaps even oil on the top of the Abbe
to worry about. And it’s not just any oil. It has to be a special type, microscope “immersion oil.”
(This is a fairly inert, “non-drying” liquid with optical properties very similar to glass.)
Second, we have already shown that the Abbe condenser starts to fail (optically) above NA 0.65,
so that 100x/1.25 objective is not ever going to function at NA 1.25 if used with an Abbe. If the
Abbe is used “dry” (without any immersion oil between its top and the underside of the slide) then
the useful NA of the 100x objective will be around NA 0.9 or so -- and not a whole lot better even
if the Abbe is “oiled” to the slide. (This is because the Abbe condenser iris typically must be closed
down a bit to help improve the image contrast and reduce “glare.”) Thus the usable resolution with
one of these objectives (when used with an Abbe condenser) is only slightly better than with a 40x
objective (especially with careless setup, as is all too common).
Third, oil immersion objectives typically have a very limited “working distance.” This means that
for an object covered with a coverglass (e.g., typical mounted microscopic sample) the distance
between the top of the coverglass and the tip of the objective may be 0.01mm (.0004") or less. This
space will be reduced by and distance between the bottom of the coverglass and the object detail
to be focused. Thus, for many objects, there is simply no way to focus on the desired detail(s) when
using such an objective. 100x “oil immersion” objectives are only useful on very thin specimens.
Finally, the 100x objective only covers a very tiny area of the object at any one time, less than 1/6
of that covered by the 40x objective. Thus, for many structures, only a small part may be seen at
any one time, whereas the 40x objective may well show most or all of the same structure.
Of course, there’s also the small matter of the inevitable “mess” that comes from adding a thick
liquid to a working environment. You have to add the oil, then remember to clean it up before you
can switch back to using a “dry” objective. And if you forget (which happens) then you may be
faced with having to clean up the “dry” objective as well (usually the 40x) which may not be easy.
Still, even after having “shot holes” in most of the reasons for using one of these objectives, we
need to see how one should be used properly. After all, if its sitting there on the scope, sooner or
later the user will find an excuse for using it!

I-84
4.10 Using Oil Immersion
Regardless of whether or not the condenser top is oiled to the slide (a step commonly avoided as
it actually results in little improvement and a lot bigger mess to clean up . . . ), an “oil immersion”
objective MUST be oiled to the slide if it is to function in a useful manner. (Oil objectives may be
used dry but the image quality in the “dry” mode is usually very poor. The only time they provide
an even halfway decent image “dry” is when the tip is nearly in contact with the coverglass and
the condenser iris is closed down. And while it is also possible to employ water as an immersion
fluid, provided the objective tip is clean and the water is removed quickly after use, even this often
results in a somewhat degraded image. Also, note that water, being less viscous than oil and
also having a higher “vapor pressure” is much more likely to seep into the guts of an
expensive lens if it is not perfectly sealed. And once water gets inside, you have a real
problem. For this reason, the use of water is suggested only with an older, expendable
objective or only for very brief use.
The process of “oiling” an objective is a bit of an art -- particularly so when an inexpensive
objective is used. This is because such objectives (and many older ones) usually have a very tiny
front element which is slightly recessed into the metal tip for protection. Under such conditions
it can be rather difficult to achieve complete oil contact between the front element and the
coverglass on the slide as the small recess tends to entrap a tiny amount of air. Such air entrapment
(or “bubble”) results in an optically-defective path, and hence a very poor image, if any at all.
Now, some objective designs seem more prone to this problem then others, and many of the more
costly oil immersion objectives are designed with nearly flat front surfaces and larger exposed
glass areas. Both of these steps tend to facilitate the “wetting” of the objective’s front element by
drawing oil across it. (A quick look will show you which type of front your objective has.)
For troublesome objectives it may well be necessary to first remove the objective from the
microscope, to stand it one its end (tip up) and to then place a small drop of immersion oil directly
on the front lens, being careful to avoid any air bubbles in the process. Excess immersion oil may
then be carefully wiped off, leaving a “wet” glass surface. Then the objective is then reinstalled
on the microscope. (It is also possible to “lift” a clean drop of oil onto the objective tip, rolling it
in from one side slowly, so as to avoid trapping any air. Use a toothpick or a soft, clean brush.)
Insofar as the slide is concerned, the slide is positioned roughly as desired on the stage, with the
oil objective moved at least partway aside. A small drop of bubble-free immersion oils then
deposited on the coverglass, approximately above the suspected area of interest. The oil objective
is then slowly rotated back into position, noting its height above the coverglass as it is moved. (It
should be nearly in contact with the coverglass when finally in position, but this can be difficult
to judge on early attempts.) Rotating the objective minimizes the likelihood of air entrapment.
As the objective moves into place, the oil on the coverglass should flow up and across the tip of
the objective, resulting in complete, bubble-free oil contact. (Note that it is only necessary for the
oil to completely cover the lens element on the oil objective, not the entire front. The more oil you
apply, the more there will be to clean up later . . . ) Although it may take a bit of practice to
accomplish, eventually the required oil path should be established and the process can advance to
the next steps.

I-85
Because of the very abbreviated “working distance,” focusing oil immersion objectives requires
more care than do the other objectives. This is less of a concern nowadays since nearly all such
objectives have spring-loaded (“retractable”) tips, to prevent damage to the specimen and/or
objective. (However, some old objectives are non-retractable and can even smash right through a
glass slide if use carelessly!)
The general practice for such older lenses is to initially position them so that the objective’s tip is
nearly in contact with the coverglass, and then to focus “upward” vary slowly, while viewing
through the microscope.
With all these high-magnification, high-NA objectives the image has a tendency to suddenly “pop”
into near-focus and then to disappear just as quickly as the proper focus point is passed. Thus
focusing slowly is necessary to avoid simply passing through the tiny focal region so quickly that
the image is completely missed.
To help in locating the focus, closing the condenser iris about halfway may aid in focusing by
increasing image contrast and slightly improving the “depth-of-focus” -- just don’t forget to open
it up again later! (In general, it’s best to simply leave the condenser iris nearly fully-open with the
100x objective, unless this creates a “glare” problem on your scope.)
Finally, when you are done using an oil immersion objective, be careful not to rotate the 40x (or
other) objective right back into the immersion oil (this is a common oversight and, with many
“dry” objectives, and getting them properly cleaned is not always easy! (Note: If your 40x
objective should suddenly stop giving you good images, oil on the front element of that
objective is one of the first things to check for.)
Modern synthetic immersion oil, unlike the older “natural” types, may be left on the front of the
oil objective for a considerable period without harm. Thus, it is not necessary to clean off the oil
objective thoroughly after each and every use -- simply wiping off any excess oil, carefully, is
about all that’s normally required. However, if the objective is not likely to be used again for an
extended period (like, weeks), then it may be best to remove the oil (again, carefully) using a soft,
clean cotton cloth or applicator, moistened slightly with a non-aggressive solvent, such as Naphtha
(lighter fluid). (Aggressive solvents, like Xylene (“Xylol”) should be avoided, unless you just
happen to be using genuine Cedarwood oil for immersion -- which you should NOT be doing.)

And so, now . . .

You have now begun to master the art of using your microscope and all its objectives !

E N JO Y U S IN G IT !!!

*************************

I-86
Notes

I-87
Notes

I-88
 Appendix I
Glossary of Microscopy Terms
Abbe -- A simple, inexpensive two-lens condenser design, but the most common type.
Aberration -- A defect in an optical system that is predictable from its design.
Achromatic -- Means “without coloring”, but should be interpreted as “without false coloring”.
Anoptral -- An optical contrasting system similar to Phase Contrast, but not as popular.
Aperture -- Optically, the maximum opening for the imaging system optical path.
Aplanatic -- An optical corrected for “spherical aberration”.
Apo / Apochromat -- Truly, “without coloring”. “True-to-life” color, but at a high price!
Balsam -- see Canada Balsam.
Bertrand Lens -- A special lens in a microscope that permits viewing the objective rear aperture.
Canada Balsam -- An old, natural mountant. Tends to “yellow” with age.
CDM -- Chromatic Difference of Magnification, a measure of optical “color compensation” (p.29).
Chromatic Aberration -- This is “false color” -- typically blue or orange color fringes near edges.
Condenser -- An optical component designed to maximize the usable aperture of an objective.
Coverglass -- A thin piece of optical glass which protects a specimen. basically a small “window”.
Coverslip -- see Coverglass.
Critical Illumination -- A classic form of microscope illumination where the actual light source
is imaged on the object. (Compare this to Kohler Illumination, below.)

Darkfield -- A form of object illumination where the object is strongly side-lighted (only).
Diatom --- A microscopic plant form which features a high-detailed hard silicon shell.
Diffuser -- A component which scatters light in many directions, creating a “softer” light.
Epi-Illumination -- Illumination of an object via the viewing optics (e.g., Reflected light).
Epi-fluorescence -- Epi-Illumination, but typically using UV light, looking for fluorescence.
Field Iris -- The variable aperture on an illuminator light port which is used to control the diameter
of the illuminator’s output beam.
Field Stop -- An aperture stop in an eyepiece which creates a sharp outline for the field-of-view.
Fluorite -- A natural mineral with high-desirable optical properties. Replaced by new glass types.
Fluor -- A name now often applied to objectives using new, superior optical glass types.
Focal Plane -- The plane location in an optical system were a “real”image is formed.

Glycerin -- A clear liquid, relatively harmless, often used in microscopy.

Glycerin Jelly -- An archaic mountant, often used for very delicate objects. Easy to make.

Hoffman Modulation Contrast -- A patented contrasting system providing a “3D-like” image.

I-89
Notes

I-90
Immersion Oil -- A special liquid with optical properties very similar to glass. Non-drying.
Index of Refraction -- see Refractive Index.
Infinity Optics -- An optical system which allows significant separation without ruining image.
Iris Diaphragm -- A mechanical device which produces a variable opening via a set of leaves.

Kohler Illumination -- An illumination system which images the actual light source in the “rear
aperture” (also, “rear focal plane”) of the objective.
Light Source -- Source of illumination. Typically from an electric lamp of some type, in which
case this term refers to the glowing “filament” of the lamp.
Magnification -- A measure of the degree of visual enlargement of an object, versus the size seen
with the unaided eye, at a standard viewing distance of 10 inches.
Naphtha -- Common “lighter fluid” and a very useful, often overlooked solvent in microscopy.
Nomarski DIC -- A contrasting method based on complex optics. Provides “optical sectioning”.
Nosepiece -- A rotating holder for objectives which provides accurate, repeatable positioning.
Numerical Aperture (NA) -- A measure of “Resolving Power”. Usually marked on an objective.
Technically, “the sine of the half-angle of the light cone accepted by the objective”.
Objective -- A lens designed to work in close proximity to an object, under special conditions.
Object Plane -- That portion of the object )on hte stage) which is in focus.
Ocular -- see Eyepiece.
Okular -- German for “ocular,” or eyepiece.
Oil Immersion -- An objective (or condenser) that works through a thin layer of immersion oil.
Optical Tube Length -- The distance from the rear of the objective to the intermediate image.
Phase Contrast -- A contrasting method that enhances optical “phase shifts” (produced by the
specimen) using special objectives.
Plan -- A prefix denoting an apparent flat field of view, basically in-focus, “edge-to-edge”.
Refractive Index (RI) -- A measure of a optics ability to “bend” (or “deviate”) light.
Rear Aperture -- The “back focal plane” or “rear focal plane” of an objective.
Resolution -- The ability to see fine detail. Can be calculated from the objective’s NA, and
is approximately: 0.32 microns divided by the objective NA. [ = 0.32u./NA ]
Resolving Power -- see “Resolution”, above.

Spherical Aberration -- An optical defect where light rays to not come to a sharp focus.

Tube Length -- The distance from the objective seat (nosepiece face) to the top of the eye tube(s).
(More specifically, this is the “Mechanical” tube length of the microsocpe)
UV -- Ultra-violet light. Short-wave, high-energy light used for fluorescence. Requires precautions.
Xylene -- A chemical solvent historically used in microscopy. Requires precautions.
Xylol -- Xylene.

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Notes

I-92
 Appendix II
Suggested Reading
Allen, R.M., The Microscope, D. Van Nostrand Co., Inc. 286pp.,1940.

Belling, The Use of the Microscope, McGraw-Hill Book Co., Inc., 315pp., 1930.

Bradbury, S., The Evolution of the Microscope, Pergamon Press, Ltd., 357pp., 1967.

Gage, Simon H., The Microscope, 17th ed., Comstock Publishing Co., Inc., 617pp., 1943.

Hartley, W.G., Hartley’s Microscopy, Senecio Publ. Co., Ltd., 220pp., 1979.
(ibid)., The Light Microscope, Its Development and Use, Senecio Publ. Co., 360pp., 1993.

James, J., and Tanke, H.J, Biomedical Light Microscopy, Klewer Academic Publ., 192pp.,1991.

Needham, George H., The Practical Use of the Microscope, Ch. H. Thomas, Inc., 491pp., 1958.
(ibid)., The Microscope, A Practical Guide, Charles H. Thomas, Inc.,115pp., 1968.

Smith, Warren G., Modern Optical Engineering, McGraw-Hill Book Co., Inc. 476pp.,1966.

Introductory Reading
Corrington, Julian D., Exploring With Your Microscope, McGraw-Hill, 229pp., 1957.

Grave’, Eric V., Using the Microscope, Dover Publications, Inc., 195pp., 1984.

Johnson, G., and Bleifield, M., Hunting with the Microscope, Arco Publ. Inc., 197pp., 1974.

Munoz, F.J, and Charipper, H.A., The Microscope and Its use, Chem. Pub. Co., 334pp., 1943.

Nachtgill, Werner, Exploring with the Microsocpe, Sterling Publishing Co, Inc., 160pp., 1995.

Stelhi, Georg, The Microscope and How to Use It, Dover Publications, Inc., 157pp., 1960.

Stevens, M. Brian, The Microscope on a Budget, Logical Image Research, 247pp, 1993.

I-93
 Combined Index
(Note: Index format is, Volume (I or II) - page number.)
Aperture, Rear: Illumination:
(see, “Rear Aperture”) Basic Setup I-74
Correcting Faults I-60
Binocular (Body):
Field Iris I-66
Adjustment I-77
Kohler (vs Critical) I-48, (Setup) I-49
Body (Types) I-75, I-77
Kohler (Simplified) I-50, (Setup) I-67
Condenser(s): Proper...? I-56
Abbe I-74, (other) I-55 “Real World” I-45, (and...) I-63
and Illumination I-47 Viewing Faults I-61
Darkfield II-4, Verifying I-58
Higher Performance I-52
Image:
Phase Contrast II-15
Critical Factors I-31
Special-Purpose I-54
Faults (Summary) I-30
Types (Summary) I-53
“Optimizing” I-17
Contrast (Methods):
Immersion:
Brightfield
Darkfield (Setup) II-11
Darkfield II-4
Objectives (for)
Jamin-Lebedeff II-31
Oil (Observation) I-84, (Using) I-85
Nikon Color Phase II-30
Nomarski DIC II-31 Index, Refractive:
Oblique II-19 (see, “Refractive Index”)
Phase Contrast II-15
Infinity Optics:
Pseudo-Phase/COL II-24
Infinity Systems I-41
Rheinberg Color II-21
Magnification I-42
Coverglass(es): Tube Lens I-41
Thickness I-33, II-7
Microscope(s):
Darkfield: Classic/Vintage (Types) I-11
Abbe, Patch Stop II-3 “Compound” I-73
Condensers (Types) II-4 Differences (Types) I-5
Limitations (Summary) II-1 Features (Summary) I-7, I-13
Performance Factors II-8 Lab (Types) I- 4, (vs. Stereo) I-3
Problems (Summary) II-12 Lab (Differences, Summary) I-5
Setup (dry) II-9, (oil) II-11 Limitations (Summary) I-15
Vs. Phase Contrast II-17 “Optimizing” I-17
Problems (Cost-cutting) I-9
Eyepiece(s):
Research (Types) I-10
Choices (“Real World“) I-43
“Things to Avoid” I-8
Compensation I-30
Types (Summary) I-4
“Mix-n-Match” I-35
Vintage I-11
Parameters (Summary) I-28
Weaknesses I-9

II-41
Numerical Aperture (NA): Refractive Index:
“and Condenser” I-47 Values (Summary) I-32
See also, “Resolution”.
Rheinberg:
Objective(s): Color Filter Specs II-22
Choices (“Real World”) I-42
Resolution:
Darkfield II-7
Limits (Summary) I-15
Microscope I-25
“Mix-n-Match” I-35 Setup (Adjustment):
Parameters (Summary) I-26, I-27 Binocular Body I-77
Phase Contrast II-15 Condenser (Abbe) I-75
Darkfield (dry) II-9, (oil) II-11
Oblique (Illumination):
Illumination (Basic) I-74
Disk (for) II-20
Phase Contrast (Summary) II-16
Observation (Use):
Testing (Checking):
Depth Perceptopn I-3
(see, “Verifying”.)
Low-Power I-78
High Power I-82 Tube Length:
Medium Power I-80 160 vs 170mm I-38
Oil Immersion I-84 Infinity Optics I-41
“Mix-n-Match” I-36
Optics:
“and...Microscope” I-24 Verifying (Testing/ Checking):
“Real World” I-23 Auxiliary Lens I-19
Condenser I-18, I-20
Parfocal Distance:
Eyepieces I-17
Adaptors I-40
Illumination I-18, (Lamp) I-18
Definition I-39, (Table of) I-39
Kohler Illum. I-67,I-68
Phase Contrast: Objective(s) I-19, I-20
Condensers II-15
Working Distance:
Objectives (Summaty) II-14, II-15
Long (Stereo) I-4
Optics (for) II-13
Stereo microscope I-3
Setup (Summary) II-16
Vs. Darkfield II-17
Pseudo Phase (Contrast):
Disk (for) II-26
Disk (COL) II-28
Setup (Summary) II-27
Rear Aperture:
Illumination faults (Summary) I-30
Views (for Contrast Methods) II-36

II-42