EXPERIMENT VII: THE MICROSCOPE

Objectives:
• Magnification, resolving power, contrast, field of view, parfocal, working distance
• Recognize and give the function of the parts of a light microscope
• How to care for a compound light microscope

What is a microscope?

A microscope (from the Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see")
is an instrument for viewing objects that are too small to be seen by the naked or unaided eye
(Figure 1). There are many types of microscopes, the most common and first to be invented is
the optical microscope which uses light to image the sample. Other major types of microscopes
are the electron microscope (both the transmission electron microscope and the scanning
electron microscope) and the various types of scanning probe microscope. In this lab session
we are going to focus on light microscope. Other types are beyond the scope of this lecture.

Figure 1. A typical binocular light microscope

The light microscope, so called because it employs visible light to detect small objects, is
probably the most well-known and well-used research tool in biology.
History of the Microscope
In 1590, Zaccharias Janssen and his son Hans, while experimenting with several lenses in a tube,
discovered that nearby objects appeared greatly enlarged. That was the forerunner of the
compound microscope. In 1653, Antonie Philips van Leeuwenhoek, the father of microscope,
used small glasses to magnify the threads of clothes (Figure 2). Robert Hooke, the English father
of microscopy, re-confirmed Anton van Leeuwenhoek's discoveries of the existence of tiny
living organisms in a drop of water. Finally, Charles Spencer, invented the marvelous
microscopes in 19th century.

1
Figure 2. The illustration of van Leeuwenhoek's microscopes in 1756.

Figure 3. Parts of a typical light microscope

Parts of the Microscope
There are important features of the microscope. Before learning them, it is important to know
the functions of parts of a microscope (Figure 3).

2
1. Ocular lens (Eyepiece): It is the lens system you look through (Figure 4). Depending on the
number of ocular lenses the microscopes are called as monocular (possesses 1 ocular lens) or
binocular (posseses 2 ocular lenses). Each ocular lens has its own magnification (10 to 20X).

Figure 4. 10X ocular lenses

2. Body Tube: It connects the eyepiece to the objective lenses. The light travels from objective
lens to ocular through body tube (Figure 5).

Figure 5. body tube connecting eyepiece and objectives

3. Objective lenses: It is a set of three or four lenses mounted on a rotating nosepiece attached
to bottom of body tube (Figure 6). They gather light from the specimen and projects it into the
body tube. Usually you will find 3 or 4 objective lenses on a microscope. They almost always
consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece
lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X (Figure 7). The
shortest lens is the lowest power, the longest one is the lens with the greatest power. All
quality microscopes have achromatic, parcentered, parfocal lenses.

Achromatic: An achromatic lens is able to transmit light without separating it into colors.
Parcentered: you have your speciman fixed under the microscope so it is difectly in the center
of the x and y axis. This way, when you turn the objective, the speciman will stay perfectly in
the center.
Parfocal: A term applied to objectives and eyepieces when practically no change in focus has to
be made when one magnification power is substituted for another.

3
Figure 6. Objective lenses and turret (nosepiece)

Figure 7. Total magnifications of microscope with different objectives

4. Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and
can be rotated to easily change power (Figure 6).
5. Stage: The flat platform where you place your slides (Figure 8). Stage clips hold the slides in
place. If your microscope has a mechanical stage, you will be able to move the slide around by
turning two knobs. One moves it left and right, the other moves it up and down.

Figure 8. The stage of a microscope

4
6. Substage conderser lens: The purpose of the condenser lens is to focus the light onto the
specimen. It is located immediately under stage (Figure 9). Condenser lenses are most useful at
the highest powers (400X and above). Microscopes with in stage condenser lenses render a
sharper image than those with no lens (at 400X).

Figure 9. The direction of the light passing through condenser lens and diagrams

8. Illuminator (Light source): A steady light source (110 volts) used in place of a mirror. If your
microscope has a mirror, it is used to reflect light from an external light source up through the
bottom of the stage.
9. Base and Arm: Arm supports the tube and connects it to the base. The base is the bottom of
the microscope, used for support.

HOW TO FOCUS THE IMAGE?

In order to get a sharp and clear image of the specimen being viewed the objective lens should
be put at a specified distance from the object. This is the WORKING DISTANCE which is a
property of the lens system and is constant for any objective
For this purpose there two more parts of the microscope which may have the one of most
important functions (Figure 10):

10. Coarse Focus Adjustment Knob: It raises or lowers the body tube or the stage to focus the
optics on the specimen. It facilitates a relatively large movement.

5
USE THE COARSE ADJUSTMENT ONLY WITH SCANNING (4X) AND LOW (10X) POWER
OBJECTIVES!!!
NEVER USE IT WITH HIGH POWER (40X) OBJECTIVE!!!!!

11. Fine Focus Adjustment Knob: It changes the specimen to objective distance very
slightly. It is used for all focusing of the 40 X objective.

Figure 10. The location of Coarse focus and Fine focus knobs on a microscope
Features of the Microscope
The quality of the microscope depends of 3 important features:
1. Magnification
2. Resolution
3. Contrast

1. Magnification: It is the degree to which the image

of a specimen is enlarged (Figure 11). The
magnification on a microscope must be adjusted
carefully in proportion to distance. The higher the
magnification, the closer the lens must be positioned
to the object being observed. Most microscopes
allow for adjustment of the lens-object distance, as well as providing preset
default positions that place the higher magnification lenses closer to the slide.

6
The magnification of a microscope is measured by placing an object of known length, such as a
ruler, beneath the lens and measuring the degree to which the microscope enlarges the image.
You can use a similar procedure to get an idea of the scale of any magnification by putting a
ruler or another familiar object, like a dime or paperclip, underneath the lens with the object on
the slide. Looking through the microscope, you can compare the observed object with the
relative size of the ruler.
The magnification is adjustable on both the eyepieces and lenses of most microscopes. You can
check the lens extensions of your microscope to determine the magnification, which is usually
printed on the casing of the lens extension. The most common lens magnifications for typical
laboratory microscopes are 4x, 10x and 40x, although alternatives of weaker and stronger
magnification exist.

7
Figure 11. Change of field of view under different magnification

8
When using a light microscope it is best to start out with the lowest magnification, get your
specimen in focus, and then move up to the higher magnifications one at a time. This is the
easiest way to ensure that you will be able to focus in on your object quickly. At 400x
magnification you will be able to see bacteria, blood cells and protozoans swimming around. At
1000x magnification you will be able to see these same items, but you will be able to see them
even closer up. Below is a list of your field of view at different magnifications. Field of view is
how much of your specimen or object you will be able to see through the microscope.
At 40x magnification you will be able to see 5mm.
At 100x magnification you will be able to see 2mm.
At 400x magnification you will be able to see 0.45mm, or 450 microns.
At 1000x magnification you will be able to see 0.180mm, or 180 microns.

Field of view: Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see
when looking into a microscope. As the power gets greater, the field of view gets smaller. You
can measure this by placing a clear metric ruler on the stage and counting the millimeters from
one side to the other.
2.Resolution: It determines how well specimen detail is preserved during the magnification
process. In other words, resolution is the ability to tell two points apart as separate points.
E.g. If the resolving power of your microscope is 2μm that means two points that are 2μm apart
can be seen as separate points. If they are closer together than that, they will blend together
into one point (Figure 12).

Figure 12. Different resolving powers of the microscopes. (From left to right resolving power is
decreasing).
The resolving power of a microscope depends on
a) Numerical aperture of the lens system: It is a measure of ability of microscope objectives to
gather light and resolve specimen detail at a fixed object distance.
b) Wavelength of light (the color): Microscopes are often equipped with blue filters because
blue light has the shortest wavelength in the visible spectrum. It means that if all the factors are
equal, resolving power is increased by reducing the wavelength of light used.

9
3.Contrast: It is the ability to see particular detail against the background, and is based on the
differential absorption of light by parts of the specimen (Figure 13). Since most of the biological
staff are transparent (except for pigmented organelles and tissues), the contrast ability of the
microscope becomes inadequate. For that purpose, the transparent specimen could be stained
with some special dyes.

Figure 13. Different views of the specimen as a result of different contrasts

Properties of the objective lenses

In general, the objective working distance decreases as the magnification and numerical
aperture both increase.
In general, the objective working distance decreases as the magnification and numerical
aperture both increase.

Care of the Microscope

The microscope should be sited in a permanent position!!!

It should be carried in upright position with both hands!!!

Never touch the lenses of the microscope with your hands!!!

Always start working with scanning power objective (4X)!!!

10
Use coarse focus knob only with 4X and 10X objectives. Never use it with 40X objectives or
more. Otherwise, you broke the lenses!!!

Use coarse focus knob to find the image. In order to make the image clearer you have to use
fine focus knob!!!

Do not allow liquids or dyes to get on microscope objectives!!!

Whichever objective you study, after you finish your work bring the objective to 4X and lower
the stage to the bottom and then take out the slide from the microscope!!!

The lenses should be cleaned with lens paper!!!

When not used it should be protected by a fitted plastic cover!!!

OIL OBJECTIVE LENSES (100X) SHOULD NEVER BE ALLOWED WITHOUT IMMERSION OIL!!!

DO NOT USE OIL IMMERSION OBJECTIVE (100X) UNLESS OTHERWISE REQUIRED!!!

Laboratory Instructions:
Observe and visualize the pre-made slides handed to you by your instructor under the light
microscope. Draw the different features of each cell and describe.

11