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CAS NS 2A- General

Zoology

This is a property of
PRESIDENT RAMON MAGSAYSAY STATE UNIVERSITY
NOT FOR SALE

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General Zoology

Chapter 2

The Microscope

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Chapter 2

The Microscope
Introduction

“Micro” refers to tiny, “scope” refers to view or look at. Microscopes are tools used
to enlarge images of small objects so as they can be studied. The process of using a microscope
in investigating or studying microorganism is known as microscopy. It uses a lens or a
combination of lenses to produce highly magnified images of small specimens or objects. The
compound light microscope is an instrument containing two lenses, which magnifies, and a
variety of knobs to resolve (focus) the picture. Because it uses more than one lens, it is
sometimes called the compound microscope in addition to being referred to as being a light
microscope.

In this module, students will more about the types, parts and function as well as the
proper handling and use of microscope.

Specific Objectives

At the end of this chapter, the students should be able to:

1. Identify the parts and functions of Microscope
2. Learn how to use the microscope
3. Appreciate the application and contribution of microscope in the study of life
science.

Duration

Chapter 1: The Microscope = 3 hours

(1 hour discussion; 2 hours
assessment)

Lesson Proper

The Development of Microscopes

Some of the very first magnifiers used water. There are records from 1000 BC of
people using blown glass globes filled with water to magnify things. In ancient times, people
noticed that tiny objects and writing could be seen enlarged and seen more clearly through a
round glass container filled with water.

Magnifiers are made of curved, clear pieces of glass, or plastic called lenses. Lenses
make objects look bigger by bending light rays. All light rays passing through the lens bend
toward a point called the focus. After passing through the focus, the light rays form a clear,
sharp image (likeness) of the object.

 1590 – Johannes and Zacharias Janssen (father and son) from Holland have developed
the compound microscope. They were just ordinary lens makers.

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 1624 – Italian astronomer Galileo Galilei develop the microscope and telescope
 1656 – A German mathematician Kircher made a microscope in which he saw “small
worms” when he studies plaque.
 1661 – an Italian physician Marcelo Malphigi was the first to use microscope in his
anatomical studies. His device showed that trachea (windpipe) leads to two bronchi.
This, however, was not recognized until after 200 years.
 1674 – a Dutch merchant and microscopist Anton Van Leeuwenhoek devised a
microscope with a magnification of 200-300X. He started observing microorganisms
from seawater, rainwater, teeth scrapings, fermented foods, and others. The moving
organisms he saw were named “animacules”. He submitted all his observations to the
Royal Society of London. He considered as the one who open the door to the “invisible
world of microorganisms.

Types of Microscope

The majority of microscopes are called light (bright field) microscopes since they rely
on light to observe the magnified image of a specimen or object.

Within this category are two main categories – (1) compound (high power
microscopes), and (2) stereo or dissecting (low power microscopes).

1. Compound Microscope
This is the most common type of microscope. It
can also be referred to as a biological or research
microscope. The compound microscope is what many
refer to as a high power microscope. The magnification
(power) can have a range from about 40x to 1000x and
some can go up to 1500x or 2000x. Much serious work
of a compound microscope is done at 400x to 500x.

Compound refers to the fact that in order to

enlarge an image, a single light path passes through a
series of lenses in a line where each lens magnifies the
image over the previous one. In other words, one light
path with multiple lenses equals a compound
microscope. The image is seen by the observer as if it
were only 10” (250mm) from your eye. In the standard
form the lenses consist of an objective lens (closest to the Figure 2.1.Compound Microscope
object or specimen) and an eyepiece lens (closest to the (Source: https://www.indiamart.com/proddetail/compound-
observers’ eye) and a means of adjusting the focus and microscope-18757128730.html)

position of the specimen or object.

In addition, a compound microscope uses light (reflected from a mirror, from indirect
sunlight, from desk lamps or other interior light sources, or from built-in lamps) to illuminate
the specimen or object so that you can see it with your eye. The objective lens usually consists
of three or four lenses (sometimes even five) on a rotating nosepiece (turret) so that the power
can be changed. The image produced at the eye is two dimensional (2-D) and usually reversed
and upside down. The most used light method is trans-illumination (light projected from below

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to pass through the specimen). At 400x much detail can be seen at the cellular level of
biological specimens. Learning about cells and microorganisms is both educational and
important for medical and science applications.

Parts and Functions of a Microscope

PARTS FUNCTIONS
The lens found in top where you look through. Usually have 10x,
Eyepiece
12x, or 15x magnification power
Tube Connects the eyepiece lens to the objective lens
Supports the tube and connects it to the base. It used along with the
Arm
base in carrying a microscope
Base The bottom of the microscope. Used to support the entire microscope
Illuminator A steady light source used in place a mirror
A flat platform where you place the slide. Stage Clips holds the slide
Stage
in place
Revolving The parts that holds two or more objectives. It can be rotate to change
Nosepiece or the magnification easily.
Turret
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.
Objective lenses
The shortest lens is the lowest power, the longest one is the lens with
the greatest power. The high power objective lenses are retractable
(i.e. 40XR). This means that if they hit a slide, the end of the lens
will push in (spring loaded) thereby protecting the lens and the slide.
This is an adjustment that determines how close the objective lens
can get to the slide. It is set at the factory and keeps students from
Rack Stop cranking the high power objective lens down into the slide and
breaking
things.
Many microscopes have a rotating disk under the stage. This
diaphragm has different sized holes and is used to vary the intensity
and size of the cone of light that is projected upward into the slide.
Diaphragm There is no set rule regarding which setting to use for a particular
power. Rather, the setting is a function of the transparency of the
specimen, the degree of contrast you desire and the particular
objective lens in use.
This is used to focus the microscope. It is always used first, and it is
Coarse Adjustment
used only with the low
Knob
power objective.
This is used to focus the microscope. It is used with the high- power
Fine Adjustment
objective to bring the
Knob
specimen into better focus.

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2. Stereo Microscope
Stereo microscopes are the second most common type of microscope. They can also be
referred to as dissecting, dissection, or inspection microscopes. The stereo microscope is what
many refer to as a low power microscope.

Magnification (power) can have a range from about 10x to 80x with magnification in
the 10x to 40x the most popular. Also, zoom models from about 10x to 60x or so are very
convenient. Low power is used for examining larger sized items like insect parts, plant and
flower parts, rocks and fossils, stamps, coins, PC boards, material surfaces, hair, etc. However,
specimen slides can also be observed albeit at these lower powers.

Figure 2.2. Stereo Microscope

(Source: https://www.westlab.com.au/equipment/equipment-m z/microscopes/cp-
745-microscope-led-globe-zoom-stereo)

There are two separate light paths (as opposed to a single light path in a compound
microscope) which produce a true stereo, three dimensional (3-D) image of the specimen or
object. Within the objective lens you will find two lenses (one for each path of light) side-by-
side. The optical design parameters of a stereo microscope limit its 3-D effects to low powers
only. Also in the category of low power microscopes is the single light path (like a compound
microscope) type usually referred to as a dissecting microscope.

This type is more economical than the stereo type but is very useful for examining large
sized specimens and objects. In the standard form the lenses consist of objective lenses (closest
to the object or specimen) and eyepiece lenses (closest to the observers’ eyes) and a means of
adjusting the focus (rack and pinion style) and position of the object or specimen.

In addition, a stereo microscope uses light (from desk or table lamps, indirect sunlight,
other interior light sources, or from built-in or attached lamps) to illuminate the specimen or
object so that you can see it with your eyes. The images you see are correct (upright and normal
which is the opposite of compound microscopes). Most stereo microscopes have both top and

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bottom built-in or attached illumination to handle various objects and specimens of all varieties,
shapes, and colors.

3. Electron Microscope
Ernst Ruska (1906-1988), a German engineer and academic professor, built the first
Electron Microscope in 1931, and the same principles behind his prototype still govern modern
EMs.
An electron microscope is a microscope that uses a beam of accelerated electrons as a
source of illumination. It is a special type of microscope having a high resolution of images,
able to magnify objects in nanometres, which are formed by controlled use of electrons in
vacuum captured on a phosphorescent screen. Electron microscopes use signals arising from
the interaction of an electron beam with the sample to obtain information about structure,
morphology, and composition.

Types of Electron Microscope

1. Transmission Electron Microscope (TEM)

The transmission electron microscope is used to view thin specimens through which
electrons can pass generating a projection image. The TEM is analogous in many ways to the
conventional (compound) light microscope. TEM is used, among other things, to image the
interior of cells (in thin sections), the structure of protein molecules (contrasted by metal
shadowing), the organization of molecules in viruses and cytoskeletal filaments (prepared by
the negative staining technique), and the arrangement of protein molecules in cell membranes
(by freeze-fracture).

2. Scanning Electron Microscope (SEM)

Conventional scanning electron microscopy depends on the emission of secondary

electrons from the surface of a specimen. Because of its great depth of focus, a scanning
electron microscope is the EM analog of a stereo light microscope. It provides detailed images
of the surfaces of cells and whole organisms that are not possible by TEM. It can also be used
for particle counting and size determination, and for process control. It is termed a scanning
electron microscope because the image is formed by scanning a focused electron beam onto
the surface of the specimen in a raster pattern.

Other Types of Microscopes

These are usually advanced and expensive type microscopes made for specific usages
mainly in advanced medical and research. There are many, many types but some of the more
popular types are listed below.

 Phase Contrast -- This is a microscope that uses the differences in the phase of light
transmitted or reflected by a specimen to form distinct, contrasting images of different
parts of the specimen.

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 Confocal Microscope - Unlike stereo and compound microscopes, which use regular
light for image formation, the confocal microscope uses a laser light to scan samples
that have been dyed. These samples are prepared on slides and inserted; then, with the
aid of a dichromatic mirror, the device produces a magnified image on a computer
screen. Operators can create 3-D images, as well, by assembling multiple scans. Like
the compound microscope, these microscopes offer a high degree of magnification, but
their resolution is much better. They are commonly used in cell biology and medical
applications.
 Polarizing – A microscope in which the object viewed is illuminated by polarized light
for typically analyzing the content and make-up of organic or inorganic material like
crystals, chemical microscopy, and optical mineralogy.
 Metallurgical – A microscope that is used for identification, inspection, and analysis
of different metals and alloys.
 Digital – These are a combination of a microscope and a digital camera. The more
common types are the digital cameras which can use a CCD or CMOS chip. The camera
can be integrated (built-in) with the microscope or specialized cameras (imagers) can
be purchased separately and adapted to virtually any microscope (like one pictured
below) at economical prices. With basic software provided (with the camera (or using
your own photo editing software) the user can display, save, and edit images. Some
more expensive software packages allows for a variety of image analysis useful for
medical, educational, and sophisticated research usage.
 Handheld Digital Microscopes use new technology for a miniature camera and
illuminator in one unit.

How to Use a Microscope?

1. When moving your microscope, always carry it with both hands. Grasp the arm with
one hand and place the other hand under the base for support.

Figure 2.3. Proper Carrying a Miicroscope

2. Turn the revolving nosepiece so that the lowest power objective lens is "clicked" into
position (This is also the shortest objective lens).
3. Your microscope slide should be prepared by placing a coverslip or cover glass over
the specimen. This will help protect the objective lenses if they touch the slide. Place

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the microscope slide on the stage and fasten it with the stage clips. You can push down
on the back end of the stage clip to open it.
4. Look at the objective lens and the stage from the side and turn the coarse focus knob so
that the objective lens moves downward (or the stage, if it moves, goes upward). Move
it as far as it will go without touching the slide!
5. Now, look through the eyepiece and adjust the illuminator (or mirror) and diaphragm
for the greatest amount of light.
6. Slowly turn the coarse adjustment so that the objective lens goes up (away from the
slide). Continue until the image comes into focus. Use the fine adjustment, if available,
for fine focusing. If you have a microscope with a moving stage, then turn the coarse
knob so the stage moves downward or away from the objective lens.
7. Move the microscope slide around so that the image is in the center of the field of view
and readjust the mirror, illuminator or diaphragm for the clearest image.
8. Now, you should be able to change to the next objective lens with only minimal use of
the focusing adjustment. Use the fine adjustment, if available. If you cannot focus on
your specimen, repeat steps 4 through 7 with the higher power objective lens in
place. Do not allow the objective lens to touch the slide!
9. The proper way to use a monocular microscope is to look through the eyepiece with
one eye and keep the other eye open (this helps avoid eye strain). If you have to close
one eye when looking into the microscope, it's ok. Remember, everything is upside
down and backwards. When you move the slide to the right, the image goes to the left!
10. Do not touch the glass part of the lenses with your fingers. Use only special lens paper
to clean the lenses.
11. When finished, raise the tube (or lower the stage), click the low power lens into position
and remove the slide.
12. Always keep your microscope covered when not in use. Dust is the number one enemy!

Daily Maintenance

 Inspect the microscope for damage or malfunction.

 Record any damage or malfunction in the daily microscopy maintenance sheet. Clean
the parts of the microscope with a clean cloth.
 Clean the objectives after each day’s work.
 Ensure that immersion oil residues are removed. Do not clean any part of the
microscope with xylene, which will damage the microscope and is toxic.
 Clean the objective lens with lens cleaning tissue only. Never clean lenses with alcohol,
ordinary tissues, cleaning paper, toilet paper, cotton wool or hand towels, which will
scratch the lens surface.

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 Cover the microscope with a dust cover when not in use.

 Turn off the power at the end of the day, and unplug the microscope to protect it from
a power surge.
 Do not leave lens ports uncovered; use the port cover or sealing tape.

Terms Associated with Microscope

 Magnification (power) – the magnification of a microscope is determined by

multiplying the power of the eyepiece by the power of the objective lens being used.
As an example, a 40x objective lens times 10x eyepiece = 400x.
- Another way to look at this is that the magnification is the number of times the
image is larger than the object would appear to the unaided (naked) eye. Lower
powers allow for brighter, sharper images combined with a wide field of view.
Higher powers, often for examining slide specimens, present larger but dimmer
images with a narrower field of view. When observing, always start with the lowest
power on your microscope and progress to higher powers. High powers are useless
unless the resolution is good. When the magnification is increased but the detail is
not increased (due to the limitation of the resolution), it is called “empty
magnification”.
- For a given magnification, always use the higher magnification objective when
given a choice rather than using a lower power objective with a high powered
eyepiece as the results will always be better. For example, using a 40x objective
and 10x eyepiece =400x will give higher quality views than using a 20x objective
and 20x eyepiece =400x

 Field of View – the field of view is the diameter of the circle of light that you see when
looking into a microscope and it is measured in millimeters. The lowest powers have
the widest field of view. As you increase power, the field of view gets smaller. Some
wide angle or super wide angle eyepieces increase the field of view over standard
eyepieces.

 Depth of Field – how much depth of field is a function of the objective lenses and
means the farthest and nearest points in the field of view are in simultaneous sharp
focus. Low magnification objectives have more depth of field than high magnification
objectives.

 Depth of Focus – means the farthest and nearest points in the film plane
(photomicrography) or the CCD plane (video microphotography) which are
simultaneously in focus. It is just the reverse of the depth of field, where here greater
depth of focus occurs with high magnification objectives.

 Flatness of Field – a quality describing the appearance of the field of view as being flat
from edge-to-edge.

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 Brightness – how light or dark is the image? Brightness is related to the illumination
system. It is also related to the N.A. of the objective lens where the larger the N.A., the
brighter the image at that power.

 Contrast – is the ratio of light and dark parts of a specimen or object and how well they
are defined and the main part of the microscope that affects this is the illumination
system.

 Collimation (Alignment) – this is a condition in which all optical elements are centered
on the same axis.

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Suggested Video Links

https://www.youtube.com/watch?v=-b3Eejf4rDQ

https://www.youtube.com/watch?v=SUo2fHZaZCU

Additional Resources

Burton G. R. W. & Engelkirk, Paul G. (2006). Burton’s Microbiology: For Health Sciences [
8th Edition]. 530 Walnut Street Philadelphia, PA 19106: Lippincott Williams & Wilkins Co.

Campbell, Neil A. et. Al (2005). Biology 7th Edition. Addison Wesley Longman, Inc

Sazon, Rowena R. Handbook in General Microbiology. (2010). PRMSU-CAS Biology

Department. PRMSU-Iba, Zambales

Retrieved from https://a.sellpoint.net/a/Xo1VW0NY.pdf

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Assignment

Search and study:

1. Characteristics of a Prokaryotic and Eukaryotic Cell.

2. Part and Functions of Animal Cell

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