TITLE OF LABORATORY: STUDY OF THE COMPOUND LIGHT MICROSCOPE TO UNDERSTAND THE

PARTS, USE, AND APPLICATIONS OF MICROSCOPE IN OBSERVING MICROSCOPIC BIOMOLECULES,

CELLS AND ANY MICROSCOPIC SUBSTANCES.

Aim

 To thoroughly understand the parts, use, and applications of the compound light microscope
in observing microscopic biomolecules, cells and any microscopic substances.

Introduction

Microscopes are instruments that are used in science laboratories to visualize very minute or
microscopic objects, such as cells and microorganisms, giving a contrasting image that is magnified.
Furthermore, the compound light microscope is a fundamental tool in biology for observing
microscopic structures. In this comprehensive lab, you will explore the microscope's components,
learn proper usage, and apply your knowledge to observe biomolecules and cells. Microscopes are
made up of lenses for magnification, each with its own magnification powers. Depending on the type
of lens, it will magnify the specimen according to its focal strength. Their ability to function is because
they have been constructed with special components that enable them to achieve high magnification
levels. They can view very small specimens and distinguish their structural differences. For example,
microscopes can view animal, plant cells and too microscopic bacterial cells. There are different types
of microscopes like light microscope, dark-field microscope, phase contrast microscope, electron
microscope, fluorescent microscope, etc. Microscopes are generally made up of structural parts for
holding and supporting the microscope and its components and the optical parts that are used for
magnification and viewing of the specimen images. Modern microscopes have additional electronics
and display devices. The picture below, defines the parts of a microscope and the functions they
perform to enable the visualization of microscopic specimens.

Structural parts of a microscope and their functions

When it comes to the optical parts of a microscope and their functions, it be said that, optical parts
of the microscope are used to view, magnify, and produce an image from a specimen that is placed
on a slide. These parts include:

1. Eyepiece – The eyepiece (ocular Lens) is closest to the viewer’s eye. They are located at the
top of the microscope. This part is used to look at the specimen. These lenses come in
different magnification powers from 5X to 30X, but the most common ocular lenses are of
10X or 15X magnification. They magnify the image for the second time.

2. Eyepiece tube – It’s the eyepiece holder. It carries the eyepiece just above the objective
lens. In some microscopes, such as the binoculars, the eyepiece tube is flexible and can be
rotated for maximum visualization for variance in distance. For monocular microscopes, they
are none flexible.

3. Diopter Adjustment – Diopter Adjustment is a control knob present only in the binocular
microscope that is used to change focus on one eyepiece. It is used to correct any difference
in vision and compensate for the differences in vision between the viewer’s two eyes.

4. Nose piece – A nose piece is a movable circular structure that houses all the objective
lenses. It is also called the revolving turret. It is connected to the body tube and lies just
above the stage. It can be rotated clockwise or counterclockwise to increase or decrease the
magnification. The change in magnification results due to a change in the objective lens.

5. Objective lenses – The objective lens is the lens that is closest to the specimen. They are
fitted on the nosepiece. A standard microscope has 3 to 4 objective lenses of different
magnifying powers: 4X, 10X, 40X, and 100X. The objective lenses first receive the light
transmitted from the specimen and magnify the image for the first time. Objective lenses
are color-coded and are of different sizes. Size and color depend on the power of the lens.
The smallest lens is of the lowest power, and gradually, the longest will be of the highest
power. The high power lenses i.e. 40X and 100X, are retractable, i.e., their end can be
pushed inward. In most optical microscopes, objective lenses with 100X or more
magnification are of oil immersion type.

6. The Adjustment knobs – Adjustment Knobs are the control knobs used to focus the
microscope on the specimen. These knobs are of two types;
a. Fine Adjustment Knob: Fine Adjustment Knob is used for fine adjustment. It is a smaller
knob and is used to move the stage up or down very slowly. The stage covers a very small
distance on each rotation of the fine adjustment knob. It is used to sharpen the image. It is
mostly used while viewing under high power.
b. Coarse Adjustment Knob: Coarse Adjustment Knob is used for focusing the image under
low power magnification. It is a larger knob and is used to move the stage up or down very
rapidly. The stage is raised or lowered rapidly with the help of a coarse adjustment knob.

7. Stage – This is the section in which the specimen is placed for viewing. They have stage clips
that hold the specimen slides in place. The most common stage is the mechanical stage,
which allows the control of the slides by moving the slides using the mechanical knobs on
the stage instead of moving them manually.

8. Stage Control Knobs – Stage Control Knobs are the control knobs used to move the stage
mechanically. There are two knobs; one for moving left and right and the other for moving
forward and backward. This will move the slide in the field of vision.
 9. Aperture – This is a hole in the microscope stage through which the transmitted light from
the source reaches the stage.

10. Microscopic illuminator – A microscopic illuminator is a light source. In some compound

microscopes, a mirror, which reflects the light from an external source to the sample, is
used. In other optical microscopes, different electric bulbs of low voltages are used as a
constant light source. Commonly used illuminators are tungsten-halogen lamps, 75-150W
Xenon lamps, tin-halide lamps, mercury vapor lamps, etc. The selection of types of bulbs is
based on the requirement of intensity and wavelength for illumination.

11. Condenser – These are lenses that are used to collect and focus light from the illuminator
into the specimen. They are found under the stage next to the diaphragm of the microscope.
They play a major role in ensuring clear, sharp images are produced with a high
magnification of 400X and above. The higher the magnification of the condenser, the clearer
the image. More sophisticated microscopes come with an Abbe condenser that has a high
magnification of about 1000X.

12. Diaphragm – It’s also known as the iris. It is found under the stage of the microscope, and its
primary role is to control the amount of light that reaches the specimen. It’s an adjustable
apparatus, hence controlling the light intensity and the size of the beam of light that gets to
the specimen. For high-quality microscopes, the diaphragm comes attached with an Abbe
condenser, and combined, they are able to control the light focus and light intensity that
reaches the specimen.

13. Condenser focus knob – This is a knob that moves the condenser up or down, thus
controlling the focus of light on the specimen.

14. Abbe Condenser – This condenser specially designed for high-quality microscopes makes the
condenser movable and allows very high magnification above 400X. High-quality
microscopes normally have a higher numerical aperture than objective lenses.

15. The rack stop – It controls how far the stages should go, preventing the objective lens from
getting too close to the specimen slide, which may damage the specimen. It is responsible
for preventing the specimen slide from coming too far up and hitting the objective lens.

16. Light Switch – Light Switch is an electrical control device. Light switches are used to on and
off the illuminator.

17. Brightness Adjustment – The brightness adjustment system controls the voltage supplied to
the light bulb, controlling the intensity (brightness) of the light bulb.

Apparatus/Materials

 Compound light microscope

 Microscope slides

 Cover slips

 Biomolecule and cell samples

 Microscope manuals

 Handouts with diagrams

  Ruler or callipers

 Stage micrometres

 Calculator

Standard Operating Procedure (SOP) of the Microscope

1. Carrying and Setting Up the Microscope

- Carry the microscope with both hands, one on the arm and the other on the base.

- Place the microscope on a stable surface and plug it in.

2. Adjusting the Microscope

- Turn on the microscope and adjust the lighting.

- Adjust the objective lenses and eyepieces for comfortable viewing.

3. Preparing the Sample

- Place a microscope slide on the stage and secure it with the stage clips.

- Add a cover slip if necessary.

4. Focusing the Microscope

- Focus the microscope using the coarse and fine adjustment knobs.

- Use the stage controls to centre the sample.

5. Observing and Recording

- Observe the sample and record observations.

- Take notes on the sample's appearance, size, and any notable features.

Calculating Total Magnification

To calculate total magnification;

-First identify the objective lens magnification (e.g., 4x, 10x, 40x and 100x).

- Then identify the eyepiece lens magnification (usually 10x).

- Finally, multiply the objective lens magnification by the eyepiece lens magnification to calculate the
total magnification.

Example

Total magnification = 40x objective lens x 10x eyepiece lens = 400x

Measuring Cell Length

- Use the ruler or calipers to measure the distance between two points on the microscope stage
(e.g., 1 mm).

- Convert this distance to micrometers (1 mm = 1000 μm).

- Use the microscope's stage micrometer to measure the cell length in micrometers.
- Calculate the actual cell length by dividing the measured length by the total magnification.

Example

Measured length = 50 μm, Total magnification = 400x, Actual cell length = 50 μm / 400 = 0.125 μm

Further Notes on the Microscope on the Use and Safe Operation Procedure of a Microscope

The microscope is one of the principal tools of the biologist that works by bending a beam of light
and then allow it to pass through the specimen, using a series of lenses to provide a clear image of
the specimen to the observer. In its function, a microscope magnifies a microscopic object so that it
becomes big enough to be seen by the naked eyes of observer, which without the help of the
microscope they cannot see anything of the microscopic object. Because cells are usually too small
to be seen with the naked eye. Therefore, a microscope is used as an essential tool to observe them.

In addition to magnification, microscopes provide resolution, which is the ability to distinguish two
nearby objects as separate entities. A combination of magnification and resolution in microscopy is
necessary to clearly view specimens under the microscope and appreciate their features. Without
the microscope, many of the great discoveries of biology would never have been made. The light
compound microscope, shown in the picture below, is the most commonly used in the field of
biology.

The compound microscope is used to examine cellular material and gives total magnification from
X40 up to X1000. To observe specimen on the microscope, the sample specimen needs to be
translucent. The most important feature of any lens system of the microscope is resolving power,
which is the smallest distance that separate two objects that become distinguished by the lens
system during a microscopic observation, and the resolving power that allows the two objects to be
examined as two distinct objects and not as a single entity. For example, most humans’ eyes see two
fine parallel lines as two distinct markings if they are separated by 0.1 mm. But if objects are closer
together than this distance (0.1), then the objects can be seen as a single line and the object cannot
be differentiated as two entities. Therefore, the resolving power of the human eye is 0.1 mm. When
it comes to the light microscope, it has a resolving power of about 0.0002 mm. This means that, the
light microscope can give more useful views of cells and can reveal features of some of the sub-
cellular contents of eukaryotic cells that the human naked eye cannot. Most microscopes are fitted
with four objective lenses, which magnify from X4, X10, X40 and X100. The eyepiece lens is usually
X10. To calculate total magnification of the image of an object on a microscope, the eyepiece
magnifying number should be multiplied by the power of the objective lens (magnification) as shown
in the table below.

Magnification Diameter of Field of View and Resolution

In using the microscope, it is important to know magnification and resolution. To calculate total
magnification of any specimen being viewed under the microscope, multiplication of the power of
the eyepiece (ocular lens) by the power of the objective lens being used should be done. For
example, if the eyepiece magnification is X10 and the objective lens is X40, then the magnification is
10 X 40 which gives a total magnification of X400. Although the level of magnification is almost
limitless, the resolution or resolving power of a compound microscope is not limitless. Resolution is
the ability of the microscope to discriminate or distinguish two objects which are close together as
being separate. The human eye can resolve objects about 100 µm apart. Note that: 1 µm = 1
micrometer or 1 millionth of a meter. Under ideal conditions, the compound microscope has a
resolution power of 0.2 µm, which means that objects that are closer than 0.2 µm are seen as a
single fused image. Resolving power is determined by the amount and physical properties of the
visible light that enters the microscope. In general, the greater the amount of light delivered to the
objective lens, the greater the resolution. Note that, the size of the objective lens’ aperture or
opening decreases with the increase in magnification, thereby allowing less light to enter the
objective lens. Thus, it is often necessary to increase the light intensity entering at the higher
objective magnifications so that resolution power can become greater

The Depth & Perception of a Microscope

There are two basic types of light microscope, the compound microscope and the stereoscopic
microscope. In this case, any object viewed under the microscope, has depth, length and width.
While the lens of your eye fully adjusts to focus on an object being viewed and provides you with a
three-dimensional interpretation, the lenses of a compound microscope are focused mechanically
and can only “see” objects in two dimensions, length and width. For example, if the specimen you
are examining has three layers of cells, you will only be able to focus on one cell layer at a time. In
order to perceive the relative depth of your specimen use the fine adjustment to focus through the
different planes (i.e. the three cell layers) individually to build a three-dimensional picture or
interpretation of your specimen. On the other hand, a stereoscopic microscope is used to view
whole structures of a sample specimen in three dimensions like the human eye.

Field of View of a Microscope and Estimating the Length of Specimens by Calculations

During viewing objects on a microscope, you can observe that, the object lies inside a circular field of
view, which is different for each magnification of objective lens used. Therefore, knowing the
diameter of field of view, it is easy to estimate size or length of an object seen in the field under a
microscope. Increasing magnification, results in field of view and diameter of view to get smaller
proportionately. Consequently, a critter that appears small under scanning power (X4 objective),
may appear to be large under high power objective. In this case, the actual size of a critter do not
change, only the space in which you place it for viewing does change.

To calculate, determine and estimate size or length of a specimen cell viewed on a microscope, first
know the objective lens that you are using, and after that, use diameter of field of view of a lens you
are using to calculate the size or length of the cell. Below is the table showing different diameter of
field of view for different objective lenses.

Actual Steps for Calculations of Size or Length of Cells.

a. First put your eyes on the eyepiece, Focus and observe cells that are laying along the
diameter of field of view for the objective lens that you are using.

b. Count number of cells laying along the diameter of field of view. For example, in a figure
below, the number of cells, laying along the diameters of the field of view is 8.

c. Since the objective lens that is in use in the figure is X10 with diameter of field of view 2.0
mm. Therefore, to calculate the length of the cell use the following calculations; Length =
 Diameter of Field of View/ Number of Cells Laying Along the Diameter (L of Cell = D.F.V/No
of Cells, D) =2/8 = 0.25 mm

1.0 mm = 1000 um

0.25mm = (1000) (0.25) um

= 250 um

d. Note: In the illustration above, 2.0 mm is the field of view since we are using objective X10
(with a total magnification X100). This means that, if 8 of any of the cells have to lay along or
across the diameter of field of view 2mm, then each of those cells should be 2/8 (0,25mm)
long. Since diameter of field of view depends on the power of objective used it is important
to know the diameter of field of view for each of the lens used as indicated above in the
table for diameter of field of view values.

The Oil Immersion Lens

When the oil immersion lens (X100) is used properly, it offers the ability to view objects at high
magnification of X1000, however, the objects to observe in your laboratory tasks, do not warrant its
use. As its name implies, an oil immersion lens requires a drop of immersion oil to be put in contact
between the lens and the glass slide for the lens to function effectively. Since immersion oil has the
same refractive index as glass, it prevents the scattering of light as light passes from the glass slide to
the objective lens (which is also made of glass). Poor resolution is the result if the immersion lens is
used without oil since light will be bent (and thus scattered) as it passes from the slide to air, and
then through the objective because air and glass bend light differently as a result of having different
refractive indexes.

Care of Microscope

Because microscopes are expensive instruments, therefore, they should be taken care of always
whenever using them by following general safety instructions on their use to avoid them being
damaged. Below are some of the steps to be taken into consideration when using microscopes.

a. Carry the microscope with both hands, one hand under the base, and the other on the arm.
When getting ready to put the microscope away, always return it to the low power or
scanning power setting.

b. When setting the microscope on a table, always keep it away from the edge.

c. It is generally best to clear your lab table of items that are not being used.

d. The lenses of a microscope cost almost as much as all other parts put together. Never clean
them with anything apart from lens paper. Paper towels and paper tissues will scratch them.

e. Please inform the instructor or the biology lab technician of any microscope damage or
irregularity in its operation as soon as possible. Do not return a faulty microscope without
first informing the instructor or lab technician.

f. Remember, you are responsible for the microscope when using it. So treat it with care.

Standard Operating Procedures

a. Techniques for Setting up & viewing objects with a compound light microscope should
always be followed for the safety of the microscope.
 b. Be familiar with all procedures that outline the correct use of a microscope before using it.

c. The steps for the standard operating procedure of the microscope should be observed in this
lab exercise and all other lab activities in this biology course and other courses/lab
experiences that will need a microscope.

d. To start operating the microscope, first place the scanning power (X4) objective in position
(if not already in that position) by hearing a click sound.

e. In changing from one objective to another, you should as well hear a click sound to confirm
that, the objective is set in proper position.

f. Make certain that the lenses are clean. Dirty lenses will cause a blurring or fogging of the
image. The high power and ocular lenses are the lenses that most often get dirty.

Cleaning Microscope lenses

a. To clean the lens, place a drop of lens cleaning fluid on a piece of lens paper.

b. Clean lens with a gentle circular motion, then dry with a fresh piece of lens paper.

c. Always use lens paper for cleaning! Any other material (including Kim wipes) may scratch
the lens.

Checking the Preliminary Lighting

a. Plug in the electrical cord to turn on the sub stage light. If equipped with a light switch, turn
it on too.

b. Position or set the condenser as high as it will go by turning the sub stage adjustment. This is
important as it provides for a maximum of light.

Important notice

a. When looking through the ocular or eye piece lens, you should see a white circular field of
view that is evenly illuminated.

b. Open the iris diaphragm by using the lever beneath the condenser that is positioned below
the stage of the microscope.

Placing the Slide on the Stage for Viewing

a. Make certain that the scanning power objective (4x) or the low power objective (10x) is
clicked properly in place.

b. After confirming that the scanning power objective (4x) low power objective is in place,
place now the slide on stage clip it.

Important Notice

a. If you need to scan the slide to find the location of a specimen, first use the scanning
objective (4x) with its larger field of view.

b. If you have a pretty good idea where the specimen is located on the slide it is ok to start
with the low power objective (10x).
 c. To avoid the danger of damaging high power objective lenses and their very small field of
view, never begin microscopic examinations with high power (40x) or the oil immersion
(100x) objectives.

d. Lower the stage away from the objective with the coarse adjustment and then place a
properly prepared slide (see below) on the stage and secure with the stage clips or
mechanical stage depending upon which is present on your microscope.

e. Move part of the slide with the object to be viewed directly above the brightly illuminated
sub stage condenser.

Proper Focusing Technique

a. Use the coarse adjustment knob to raise the stage until the stop is reached that will prevent
further movement of the stage.

b. Looking through the eyepiece (ocular) lens, lower the stage slowly by turning the coarse
adjustment knob away from you until the object is in focus. It should take less than a quarter
of a turn to bring the image into focus.

Technical Tips

a. When it is difficult to find a specimen to focus on (e.g. when examining amoeba), bring the
edge of the coverslip into the center of the field of view, and then try focusing on the edge.
Then search the slide for the desired specimen.

b. Reduce the light intensity to aid in the observation or viewing clear/transparent objects such
as amoeba.

c. To avoid eye strain while viewing specimens with a monocular microscope (i.e. one with only
one ocular or eyepiece), practice keeping both eyes open. Many biologists are capable of
observing a specimen and sketching it at the same time. Try it out in today’s lab.

d. Use the fine adjustment to bring the object into sharp or clear focus.

e. Adjust the amount of light with the iris diaphragm and intensity of light with the condenser
for optimum viewing. Too much or too little light adversely affects the quality of the image
viewed.

Increasing Magnification (Switching from Low to a Higher Power)

a. First, be sure that, the object that you want to view at a higher magnification is in the center
of the field of view and sharply focused under low power.

b. Then switch to high power. Watch from the side to make sure that the objective lens does
not touch the slide. Since most microscopes are par focal, it means that, little refocusing is
needed when moving from one lens to another. Only fine adjustment may be required. If
properly focused at low power, and the slide is prepared correctly (i.e. the specimen is thin
and flattened by a coverslip), you should be able to switch automatically from low power to
high power without fear of having the high power objective lens scraping or touching the
slide.
Re-Focus with the Fine Adjustment under High Power

a. Only use fine adjustment at high power. To avoid damaging the lens, never use the coarse
adjustment when the high-power objective is in place.

b. Adjust the amount and intensity of light for optimum viewing. The amount of light may need
to be increased since less light passes through the objective at higher magnification.

c. The working distance is the distance between the specimen viewed and the objective lens of
the microscope. As you increase magnification the working distance becomes less and less.
The objective will be almost touching the cover slip when properly focused at high power.

Important Notice

a. If the image viewed at the high power objective is not sharp, try to refocus, the object at the
lower power objective as it is suspected not to have been focused properly. Repeat steps of
focusing from the start.

b. Clean the objectives. Other common culprits that make image of an object not to be clear
are the high power objectives, ocular lens, cover slip, and glass slide.

General Procedures of Operating the Microscope

1. To operate safely, make sure that all backpacks, purses, etc. are off the benchtop.

2. Carry microscope by the base and arm with both hands.

3. Store with cord wrapped around microscope and the scanning objective clicked into place.

Focusing Specimens

1. Plug your microscope in to power supply and switch on illuminator.

2. Always start with the stage as low as possible and using scanning objective (4x).

3. Use the coarse knob to focus: Note: The image may be small at this magnification, but you
will not be able to find it on the higher powers without this first step. Move the mechanical
stage until your focused image is also centered.

4. Once you have focused using the scanning objective, switch to the low power objective X10.
Use the coarse knob to refocus and move the mechanical stage to re-center your image.
Again, if you have not focused on this level, you will not be able to move to the next level.

5. If the specimen is too light or too dark, try adjusting the diaphragm.

Cleanup Steps

1. Store microscope with the scanning objective in place and the stage in its lowest position.

2. Wrap cords around microscope and replace slides to original slide tray

Troubleshooting

Occasionally you may have trouble working with your microscope. Below are some of the common
problems and troubleshooting solutions.
Dark Image

a. Adjust the diaphragm while making sure that light is on and if lens are dirty, use lens paper
only to clean the objective and ocular lens. Clean inside part of ocular lens by removing it.

b. Remember the steps of focusing under scanning and then low power to focus anything
under high power.

c. If you see, only half of the viewing field and is looking like a half-moon, during microscopic
observation, it means that, maybe you do not have the objective fully clicked into place.

d. If focusing is giving you a headache, relax and try adjusting the ocular distance. Check also
that, the intensity of light is not too high or too low. Take breaks if needed.