Experiment 1 Compound Microscope

EXPERIMENT 1
COMPOUND MICROSCOPE

Structure
1.1 Introduction 1.5 Resolving Power of a
microscope
Expected Learning Outcomes
1.6 Magnification
1.2 Materials Required
1.7 Operation of a Compound
1.3 Parts of a compound
Microscope
Microscope
1.8 Precautions
1.4 Path of Light

1.1 INTRODUCTION
We will begin the Cell Biology laboratory course with exercises on handling a
compound microscope. By now, you must be familiar with the principle behind
the working of a compound microscope (Refer to Unit 2 Optical Microscopy,
Block 1 Introduction to the Cell). Some of you may have worked with a
compound microscope in your 10+2 Biology course. Although some studies
require the use of more sophisticated and powerful electron microscope which
magnifies the objects upto 500,000 times, a light microscope is sufficient for
the study of cells and tissues.

Expected Learning Outcomes

This lab exercise should help you to:
 handle a compound microscope freely and carefully;
 become familiar with the parts of a compound microscope;
 describe the resolution or resolving power of a microscope and;
 learn to draw the path of light through various lens system.

1.2 MATERIALS REQUIRED

1. Compound microscope

2. Permanent slides.

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1.3 PARTS OF A COMPOUND MICROSCOPE

Fig. 1.1: Compound microscope

A compound microscope is an optical instrument that uses a combination of

simple lenses for forming magnified images of small objects. The various parts
of a compound microscope are labeled in Fig. 1.1. The compound microscope
consists of the following parts:

1. Base: The base is made up of metal which supports the weight of the
microscope.

2. Arm: It is curved in shape and attached to the base by an inclination joint. It

supports and holds the adjustment knobs (coarse and fine control), mirror and
stage.

3. Body tube: It is a tubular body on which both lenses are mounted. The
ocular lens (eye piece) with a longer focal length is at the upper end and the
other end has the objective lens with a very short focal length. The ocular lens
is near the eye and the objective lens is just above the object.

4. Stage: A specimen stage is a rectangular platform attached to the lower

end of the arm below the objective. There is a hole at the center stage through
which light passes from the mirror and falls on the mounted slide. A pair of
clips attached to center stage holds the slide on the stage. In some
microscopes the movement of specimen stage could be controlled by knobs.

5. Diaphragm: It is present just below the stage and used for adjusting the
intensity of light that is allowed into the condenser lens.

6. Lens: Each lens system is constructed with a series of lenses, usually 8 to

10 in objectives and 2 to 3 in oculars. The closely placed elements, both in
objective and ocular lens, act as a single lens and correct any aberration that
may occur in the image. The objectives are fixed to a revolving nose-piece.
The nose-piece has provision for fixing 2 or 3 objective lenses, each with a
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Experiment 1 Compound Microscope

different magnifying power. Usually three objective lenses of 10 X (low power)

40X (high power) and 100 X (oil immersion) magnification are provided.
Generally a compound microscope has one (monocular) or two (binocular)
eyepieces.

In addition, you may observe that behind the body tube, there is provision for
two types of adjustments. There is a large knob which is a coarse control for
the vertical movement of lens tube and a fine control to bring the image of the
specimen to a sharp focus. The two adjustments are positioned on the arm of
the microscope.

A condenser lens provided just below the specimen stage can focus the light
on the specimen from any illuminated source. The condenser lens is also
formed of several lenses. The amount of light let into the condenser lens can
be controlled by an iris diaphragm placed beneath it.

Attached to the base of the microscope is a mirror which can be adjusted to

reflect the light upwards. The mirror is concave on one side and plain on the
other side. Modern compound microscope has own light source. The
illuminator (halogen bulb) is the source of light located within the base of the
microscope.

1.4 PATH OF LIGHT

The pathway of light from the condenser to the objective lens is shown in Fig.
1.2. The condenser lens can be adjusted in such a way that the light which
passes through the condenser lens converges on the specimen and then
spreads to a cone of light completely filling the objective.

Fig. 1.2: Pathway of light in Microscope

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In a compound microscope the two lenses work in tandem to produce an

image of the object. The objective located closest to the object relays real
image of the object to the eyepiece. The eyepiece in turn projects and
magnifies this real image to generate a virtual image of the object.

1.5 RESOLVING POWER OF MICROSCOPE

Assuming you place two objects close together on the stage of microscope
and if you can distinguish them as two distinct objects, then the microscope
can be said to have a high resolving power. In microscopes with low resolving
power, the image you see will appear to be that of a single object.

Let us quickly recall the meaning of resolving power of microscope.

Resolving power (d) =

Where λ is the wavelength, n is the refractive index of the medium surrounding

the specimen and α is the half angle of light entering the objective lens from
the specimen. n sin α also referred to as numerical aperture of lens (NA). The
NA of a microscope objective is a measure of its ability to gather light and
resolve the specimen detail at a fixed object distance. The resolving power is
highest when λ is smaller and α has a higher value.

In other words, resolving power is inversely proportional to wavelength. In a

light microscope, from the visible spectrum one can obtain a wavelength of 0.5
µm (0.5 x 10-6m). With the best resolution in a light microscope one can
distinguish two points which are 0.2 µm (0.2 x 10-6m) apart whereas by way of
comparison we can say that in an electron microscope it is easily possible to
resolve two points which are about 1 x 10-6m apart.

1.6 MAGNIFICATION
We earlier referred to the objective lenses of different magnifications. Similarly
the ocular lens can also be of varying magnifications. Ocular lenses will
usually have 5x 10x and 15x magnifications. Higher the magnification of ocular
lens, greater will be field diameter. You can verify this fact by changing the
ocular lens of different magnifications in the microscope. The number of times
a specimen has been magnified is obtained by multiplying the magnifying
power of ocular and objective lenses.

For instance, with an ocular lens of 10x magnification and the objective of 10x,
the specimen will be magnified 100 times. By rotating the nose piece of the
microscope you can bring the desired objective in the path of light.

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Experiment 1 Compound Microscope

1.7 OPERATION OF A COMPOUND

MICROSCOPE
Once you are familiar with the various parts of a microscope, you may start
using it. The following steps will help you to use a microscope.

1. It is important that the ocular and objective lens are clean. So prior to
using the microscope gently clean both the lenses. It is best to clean using
a lens cleaning paper and lens cleaning fluid.

2. Next rotate the revolving nose-piece and bring the low power objective
(10x) to the path of light.

3. Open the diaphragm completely.

4. While looking through the ocular of the microscope, adjust the mirror or
light source, so that the circular field that you observe gets maximum
uniform amount of light and is bright.

5. You may place any prepared slide on the specimen stage.

6. Turn the coarse adjustment knob until the objects come in view
(approximately 0.5 cm above the slide).

7. If necessary use the fine adjustment control and bring the image to sharp
focus. You may also slowly open and close the diaphragm to assess the
brightness of the field and to obtain a better contrast for the image.

8. If you need to magnify the image further, rotate the revolving nose-piece
and bring the high power objective to the path of the light. If the
microscope is parfocal you should be able to see the object clearly and
magnified. Otherwise you may use the fine adjustment knob to bring the
image into sharp focus.

9. In case your microscope has a100X objective lens, ask your Lab
instructor or counselor to show how to use an oil immersion lens.

10. Finally, after using the microscope, turn the nose-piece to bring the low
power objective to position, remove the slide from the stage, clean the
lens if necessary and return the microscope to its box.

1.8 PRECAUTIONS
1. Always keep your microscope clean and under a dust free cover.

2. Use only lens cleaning tissue papers for cleaning the lenses and never
use coarse paper or cloth as it may cause scratches in the lens.

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3. When you lift a microscope, let one hand hold the arm of the microscope
and the other support the base. Do not turn the microscope upside down
or swing it.

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