Lecture 1

The Principles of Microscopy

BMS 524 - Introduction to Confocal Microscopy and Image Analysis

Purdue University Department of Basic Medical Sciences,

School of Veterinary Medicine

J.Paul Robinson, Ph.D.

Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawleys Handbook of Confocal Microscopy Plenum Press which has been used extensively
for material and ideas to support the class.

UPDATED December 27, 1998

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 Evaluation

End of term quiz - 100% grade

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 Introduction to the Course
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications

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 Introduction to Lecture 1
Early Microscopes
Modern Microscopes
Magnification
Nature of Light
Optical Designs

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 Microscopes

Upright
Inverted
Khler Illumination
Fluorescence Illumination
"Microscope" was first coined by members of
the first "Academia dei Lincei" a scientific
society which included Galileo

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 Earliest Microscopes
1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the
first compound microscopes
1660 - Marcello Malpighi circa 1660, was one of the first great microscopists,
considered the father embryology and early histology - observed capillaries in
1660
1665 - Robert Hooke (1635-1703)- book Micrographia, published in 1665,
devised the compound microscope most famous microscopical observation
was his study of thin slices of cork. He wrote:

. . . I could exceedingly plainly perceive it to be all perforated and

porous. . . these pores, or cells, . . . were indeed the first microscopical
pores I ever saw, and perhaps, that were ever seen, for I had not met
with any Writer or Person, that had made any mention of them before
this.

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 Earliest Microscopes
1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric
merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor
city official.

Leeuwenhoek is incorrectly called "the inventor of the microscope"

Created a simple microscope that could magnify to about 275x, and
published drawings of microorganisms in 1683
Could reach magnifications of over 200x with simple ground lenses - however compound
microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke
claimed they were too difficult to use - his eyesight was poor.
Discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells,
microscopic nematodes
In 1673, Leeuwenhoek began writing letters to the Royal Society of London - published in
Philosophical Transactions of the Royal Society
In 1680 he was elected a full member of the Royal Society, joining Robert Hooke, Henry
Oldenburg, Robert Boyle, Christopher Wren

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 Secondary Microscopes
George Adams Sr. made many microscopes from about 1740-1772 but he was
predominantly just a good manufacturer not inventor (in fact it is thought he was more
than a copier!)
Simple microscopes could attain around 2 micron resolution, while the best
compound microscopes were limited to around 5 microns because of chromatic
aberration
In the 1730s a barrister names Chester More Hall observed that flint glass (newly
made glass) dispersed colors much more than crown glass (older glass). He
designed a system that used a concave lens next to a convex lens which could realign
all the colors. This was the first achromatic lens. George Bass was the lens-maker
that actually made the lenses, but he did not divulge the secret until over 20 years later
to John Dolland who copied the idea in 1759 and patented the achromatic lens.
In 1827 Giovanni Battista Amici, built high quality microscopes and introduced the
first matched achromatic microscope in 1827. He had previously (1813 designed
reflecting microscopes using curved mirrors rather than lenses. He recognized the
importance of coverslip thickness and developed the concept of water immersion

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 Lister, Abbe, Zeiss & Schott
In 1830, by Joseph Jackson Lister (father of Lord Joseph Lister) solved the
problem of Spherical Aberration - caused by light passing through different
parts of the same lens. He solved it mathematically and published this in the
Philosophical Transactions in 1830
Ernst Abbe together with Carl Zeiss published a paper in 1877 defining the
physical laws that determined resolving distance of an objective. Known as
Abbes Law
minimum resolving distance (d) is related to the wavelength of light (lambda) divided by the
Numeric Aperture, which is proportional to the angle of the light cone (theta) formed by a point on
the object, to the objective.
Abbe and Zeiss developed oil immersion systems by making oils that
matched the refractive index of glass. Thus they were able to make the a
Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to
resolve two points distanced only 0.2 microns apart (the theoretical maximum
resolution of visible light microscopes). Leitz was also making microscope at
this time.
Dr Otto Schott formulated glass lenses that color-corrected objectives and
produced the first apochromatic objectives in 1886.

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 Modern Microscopes
Early 20th Century Professor Khler
developed the method of illumination still
called Khler Illumination
Khler recognized that using shorter
wavelength light (UV) could improve
resolution

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 Khler
Khler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
Two conjugate image planes are formed
one contains an image of the specimen and the
other the filament from the light

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 Khler Illumination
condenser
eyepiece
Field iris Specimen Field stop
retina

Conjugate planes for image-forming rays

Specimen Field stop

Field iris

Conjugate planes for illuminating rays

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 Some Definitions
Absorption
When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
Refraction
Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
Diffraction
Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
Dispersion
Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow

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 Refraction

Short wavelengths are bent

more than long wavelengths

dispersion

Light is bent and the resultant colors separate (dispersion).

Red is least refracted, violet most refracted.

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 Refraction

He sees the
fish here .

But it is really here!!

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 Control

Absorption

No blue/green light
red filter

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 Light absorption

white light blue light red light green light

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 Absorption Chart
Color
Colorin
inwhite
whitelight
light Color
Colorof
oflight
lightabsorbed
absorbed
red blue green
blue red green
green red blue
yellow blue
magenta green
cyan red
black red green blue

gray pink green blue

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 The light spectrum
Wavelength ---- Frequency
Blue light
Photon as a
488 nm wave packet
short wavelength of energy
high frequency
high energy (2
times the red)
Red light
650 nm
long wavelength
low frequency
low energy

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 Magnification
An object can be focussed generally no closer than
250 mm from the eye (depending upon how old
you are!)
this is considered to be the normal viewing
distance for 1x magnification
Young people may be able to focus as close as 125
mm so they can magnify as much as 2x because
the image covers a larger part of the retina - that is
it is magnified at the place where the image is
formed

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 Magnification 1000mm

35 mm slide
24x35 mm

1000 mm
M = 35 mm = 28

p The projected image is 28 times

larger than we would see it at 250
mm from our eyes.
If we used a 10x magnifier we would have a
magnification of 280x, but we would reduce the field
of view by a factor of 10x.
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 Some Principles
Rule of thumb is is not to exceed 1,000
times the NA of the objective
Modern microscopes magnify both in the
objective and the ocular and thus are called
compound microscopes - Simple
microscopes have only a single lens

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 Basic Microscopy
Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
Structural details emerge via phase
differences and by staining of components
The edge effects (diffraction, refraction,
reflection) produce contrast and detail

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 Microscope Basics
Originally conformed to the
German DIN standard Object to
Image
Standard required the following Distance Mechanical
real image formed at a tube length = 195 mm tube length
of 160mm = 160 mm

the parfocal distance set to 45 mm

object to image distance set to 195 Focal length
mm of objective
= 45 mm
Currently we use the ISO
standard

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 The Conventional Microscope

Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm

Focal length
of objective
= 45 mm

Modified from Pawley Handbook of

Confocal Microscopy, Plenum Press

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 Upright Scope

Epi-
illumination
Source

Brightfield
Source

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 Inverted Microscope

Brightfield
Source

Epi-
illumination
Source

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 Conventional Finite Optics
with Telan system Modified from Pawley Handbook of
Confocal Microscopy, Plenum Press

Ocular
Intermediate Image

195 mm
160 mm
Telan Optics

Other optics

Objective
45 mm

Sample being imaged

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 Infinity Optics
Ocular

Primary Image Plane

The main advantage of

infinity corrected lens systems
Tube Lens is the relative insensitivity to
additional optics within the
Infinite Other optics tube length. Secondly one can
Image focus by moving the objective
Distance Other optics and not the specimen (stage)

Objective Modified from Pawley Handbook of

Confocal Microscopy, Plenum Press

Sample being imaged

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 Summary Lecture 1
Simple versus compound microscopes
Achromatic aberration
Spherical aberration
Khler illumination
Refraction, Absorption, dispersion,
diffraction
Magnification
Upright and inverted microscopes
Optical Designs - 160 mm and Infinity optics

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