MOI UNIVERSITY

SCHOOL OF SCIENCE AND AEROSPACE STUDIES

DEPARTMENT OF BIOLOGICAL SCIENCES

Lecture 2: Introduction to the Microscope

• Microscope – an instrument that produces an enlarged image of an object.
• Biologists use microscopes to study cells, cell parts, and organisms that are too small to be seen with the
naked eye.
• Microscopes magnify and show details of the image.
• Two types of microscopes:
o Light Microscope – light passes through one or more lenses to produce an enlarged image of a
specimen
o Electron Microscope – forms image of a specimen using a beam of electrons rather than light
• Some early microscopes:

• Zacharias Janssen – Dutch spectacle maker who tested several lenses in a tube and discovered that
nearby objects appeared significantly enlarged.

• Robert Hooke – used a microscope to observe a thin slice of cork. The spaces he saw reminded him of
the small rooms where monks lived, so he called them “cells,” which he used to describe the smallest

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
 units of life.
o He used microscopes with two and three lenses, but they didn’t produce very detailed images.
o Drawings made by Robert Hooke:

• Anton van Leeuwenhoek - Dutch merchant who learned how to grind lenses to make simple
microscopes (have only one lens). These produced clearer and more enlarged images than Hooke’s.
o He is considered “The Father of Microscopy” and built over 500 different microscopes.
o He was the first to discover microorganisms under a microscope by observing a drop of pond
water filled with life. He called them “tiny animalcules.”
o He also saw and studied bacteria and the blood flow through capillaries in the tail of a fish.
o Drawings made by Anton van Leeuwenhoek:

apart.

Parts of a Compound Light Microscope:

1. Body tube: Keeps the two sets of lenses a set distance
2. Rotating nosepiece: Allows one to
change between the objective lenses.
3-5. Objective lens: The second set of lenses in a
compound microscope (usually 4x, 10x, 40x).
6. Stage clips: Hold the slide in place.
7. Diaphragm: Adjusts the amount of light
that hits the slide from the light source.
8. Light source: Where light comes from to see image.
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9. Ocular lens: The first lens in a compound microscope
(usually 10x).
10. Arm
11. Stage: Where one puts the slide to view.
12. Coarse Adjustment Knob: Moves the stage up and
down very quickly.
13. Fine Adjustment Knob: Moves the stage up and
down very slowly.
14. Base
• Compound Light Microscope
o Light passes through the specimen on the slide and uses two lenses to form its image.
o Capable of two things (below) that vary in different microscopes:
▪ Magnification: a measure of how much the image is enlarged
Total magnification = (ocular lens)(objective lens being used)
[The ocular lens usually has a 10x magnification, but that can vary.]

4x objective lens = (10x)(4x) = 40 times total magnification

10x objective lens = (10x)(10x) = 100 times total magnification
40x objective lens = (10x)(40x) = 400 times total magnification

▪ Resolution: a measure of the clarity of an image; how clear the details are

The Electron Microscope

• Resolution is the limiting factor to a light microscope since the greater the magnification is, the less it is
able to resolve the image. At magnifications beyond 2,000x, the image becomes blurry, but electron
microscopes can be used at greater magnifications.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
• Characteristics of the Electron Microscope
o A beam of electrons is used to produce an enlarged image of the specimen (it does not use light).
o This electron beam and the specimen must be placed inside a vacuum chamber so that the
electrons in the beam do not bounce off gas molecules in the air.
▪ Since living things cannot survive in a vacuum, the electron microscope cannot be used to
view living cells.
o Much more powerful than light microscopes.

pollen Dust mite mitochondrion

o There are two types of electron microscopes:

1. Transmission Electron Microscope (TEM)

▪ Uses a beam of electrons transmitted through a very thinly sliced specimen. Magnets guide
the beam of electrons toward the specimen, and the image is produced to view.
▪ Magnification to 200,000 times.
2. Scanning Electron Microscope (SEM)

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
 ▪ Provides detailed 3-D images.
▪ The specimen is sprayed with a fine metal coating (it is not sliced to view as in the TEM).
▪ As the beam of electrons is passed over the specimen’s surface, the metal coating emits a
shower of electrons, and a 3-D image of the specimen’s surface is produced to view.

Rules for Using the Compound Microscope

1. ALWAYS carry the microscope with one hand holding the arm and your other hand supporting
under the base.
2. Plug in the cord and turn on the light source of the microscope.
3. Place your slide on the stage and arrange the stage clips to hold the slide in place.
o Keep the stage dry and ALWAYS make sure that your slide is dry, especially the bottom,
before putting it on the microscope.
4. Always start with the 4x objective lens (should already be on this from when the microscope was
put away). Focus this objective lens using the coarse adjustment knob.
5. Once the image is in focus, carefully swing the 10x objective lens in place and focus this objective
lens using the coarse adjustment knob.
6. Once the image is in focus, VERY carefully swing the 40x objective lens into place – BE SURE
TO NOT TOUCH THE SLIDE. Focus this objective lens using ONLY the fine adjustment knob.
o NEVER use the coarse adjustment knob while using the high power objective lens (40x).
7. Make observations and take notes as needed before preparing to put the microscope away (#8-11).
8. Lower the stage using the coarse adjustment knob.
9. Swing the objective lens back to low power (4x).
10. Turn off light source, unplug and neatly wrap cord around microscope (NOT around lenses or
light source).
11. Place protective cover over microscope before you put the microscope away.

Preparation of a Wet Mount Slide

• Wet mount slides are used to view living organisms, such as ones that need to be kept moist, and any
liquid substances.

1. Using the appropriate tool, put your specimen in the center of a clean and dry slide.
2. Add one large solid drop of water over your specimen (water should not move on slide).
3. Hold a clean and dry coverslip at a 45° angle over your specimen/water drop. Let one edge of
the coverslip touch an edge of the water drop.
4. Gently drop the rest of the coverslip into place – want to avoid getting air bubbles (the whole
slip should touch water). *Remember to keep the rest of the slide [and microscope stage] dry!*

How to Measure Under the Microscope

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
 • The micrometer (m) is the unit of measurement used to measure things under the microscope.
o One micrometer = 0.000001 meter (10-6).
• To estimate the size of each cell, use the diameter of each objective lens shown below.

The 10x objective lens has a field of view The 40x objective lens has a field of view
with a diameter of 1,500 m. with a diameter of 375 m.

Cell Cell

The size of the cell would be about 500 m. The size of the cell would be about 100 m.

Light Microscope: Principle, Types, Parts, Diagram

The evolution of the Microbiology field put to perspective the need to identify, view, observe and understand
microorganisms, including their structural morphologies and mechanisms. Microbiology’s scope is to study organisms and
minute agents that can only be examined and observed with a microscope.

Although scientifically, the first simple microscope was discovered by two Dutch scientists, Zaccharias Janssen and his
father, Hans who made spectacles, were the first to experiment with their lenses by combining lenses in a tube and observed
that the objects that were nearby, appeared closer and larger. Despite not being included as a scientific discovery, this act
paved the way for scientific evolution.

From the History of Microbiology, Antony Van Lewnehoueek an amateur Microbiologist made the first simple microscope,
that enabled him to observe the presence of tiny living organisms in pond water that appeared like dots. His simple
microscope was made up of a double convex glass lens that was held between two silver plates.

The application of microscopy in Microbiology enhanced the visualization of cells and microorganisms by magnifying their
images to make them larger.

The light microscope is also known as an optical microscope.

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What is a light microscope?

A light microscope is a biology laboratory instrument or tool, that uses visible light to detect and magnify very small
objects and enlarge them.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
 • They use lenses to focus light on the specimen, magnifying it thus producing an image. The specimen is normally
placed close to the microscopic lens.

• Microscopic magnification varies greatly depending on the types and number of lenses that make up the microscope.
Depending on the number of lenses, there are two types of microscopes i. e Simple light microscope (it has low
magnification because it uses a single lens) and the Compound light microscope (it has a higher magnification
compared to the simple microscope because it uses at least two sets of lenses, an objective lens, and an eyepiece).
The lenses are aligned in that, they can be able to bend light for efficient magnification of the image.

• The functioning of the light microscope is based on its ability to focus a beam of light through a specimen, which is
very small and transparent, to produce an image. The image is then passed through one or two lenses for
magnification for viewing. The transparency of the specimen allows easy and quick penetration of light. Specimens
can vary from bacterial to cells and other microbial particles.

Diagram of Light
Microscopes

Principle of a light microscope (optical microscope)

As mentioned earlier, light microscopes visualize an image by using a glass lens, and magnification is determined by, the
lens’s ability to bend light and focus it on the specimen, which forms an image. When a ray of light passes through one
medium into another, the ray bends at the interface causing refraction. The bending of light is determined by the refractive
index, which is a measure of how great a substance slows the speed of light. The direction and magnitude of the bending of
the light are determined by the refractive indexes of the two mediums that form the interface.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Principle of a light microscope

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
A medium with a lower refractive index such as glass to air normally speeds up the light penetration and makes light bend
away from the normal and when light is passed through a medium with a greater refractive index such as air to glass, it
normally slows down and bends towards the normal, perpendicularly to the surface.

If an object is put between these two mediums i.e between water and air, in this case, a prism, the prism will bend the light at
an angle. This is how the microscopic lenses work, they bend the light at an angle. The lens (convex) on receiving the light
rays, focuses the rays at a specific point known as the focal point (F-point). The measure of distance from the center of the
lens and the focal point is known as the focal length.

A microscope uses lenses whose strength is predetermined, in that, the strength of a lens is directly related to the focal length
i.e short focal length magnifies objects more than lenses with a long focal length.

Microscopy works strictly with a factor of resolution whereby resolution is the ability of a lens to be able to differentiate
small objects that are closely packed together. The resolution of a light microscope is determined by a numerical
aperture of its lens system and by the wavelength of the light it employs; a numerical aperture is a definition of the light
wavelengths produced when the specimen is illuminated.

A minimum distance (d) between two objects that distinguishes them to be two separate entities, determined by the
wavelengths of the light can be calculated by an Abbe equation using the wavelength of the light that illuminated the
specimen (Lambda, λ) and the numerical aperture (NA, n sin Ɵ) i.e. d=0.5 λ/n sin Ɵ

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Figure: Labeled Diagram of a Light Microscope.

Types of light microscopes (optical microscope)

With the evolved field of Microbiology, the microscopes

used to view specimens are both simple and compound light microscopes, all using lenses. The difference is simple light
microscopes use a single lens for magnification while compound lenses use two or more lenses for magnifications. This
means, that a series of lenses are placed in an order such that, one lens magnifies the image further than the initial lens.

The modern types of Light Microscopes include:

1. Bright field Light Microscope

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
 2. Phase Contrast Light Microscope

3. Dark-Field Light Microscope

4. Fluorescence Light Microscope

Brightfield Light Microscope (Compound light microscope)

This is the most basic optical Microscope used in microbiology laboratories which produces a dark image against a bright
background. Made up of two lenses, it is widely used to view plant and animal cell organelles including some parasites such
as Paramecium after staining with basic stains. Its functionality is based on being able to provide a high-resolution image,
which highly depends on the proper use of the microscope. This means that an adequate amount of light will enable
sufficient focusing of the image, to produce a quality image. It is also known as a compound light microscope.

Parts of a bright-field microscope (Compound light microscope)

It is composed of:

• Two lenses which include the objective lens and the eyepiece or ocular lens.

• Objective lens is made up of six or more glasses, which make the image clear from the object

• The condenser is mounted below the stage which focuses a beam of light onto the specimen. It can be fixed or
movable, to adjust the quality of light, but this entirely depends on the microscope.

• They are held together by a sturdy metallic curved back used as an arm and a stand at the bottom, known as
the base, of the microscope. The arm and the base hold all the parts of the microscope.

• The stage where the specimen is placed, allowing movement of the specimen around for better viewing with the
flexible knobs and it is where the light is focused on.

• Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the microscopes’ arm,
which can move the stage or the nosepiece to focus on the image. the sharpen the image clarity.

• It has a light illuminator or a mirror found at the base or on the microbes of the nosepiece.

• The nosepiece has about three to five objective lenses with different magnifying power. It can move round to any
position depending on the objective lens to focus on the image.

• An aperture diaphragm also is known as the contrast, which controls the diameter of the beam of light that passes
through the condenser, in that, when the condenser is almost closed, the light comes through to the center of the
condenser creating high contrast. But when the condenser is widely open, the image is very bright with very low
contrast.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Magnification by Bright field Microscope (Compound light microscope)

During visualization, the objective lens remains parfocal which means, when the objective lens is changed, the image still
remains in focus. The objective lens plays a major role in focusing the image on the condenser forming an enlarged clear
image within the microscope, which is then further magnified by the eyepiece to a primary image.

What is seen in the microscope as an enlarged clear image of the specimen is known as the virtual image. To calculate the
magnification, multiply the objective and eyepiece objective magnification together. The magnification is standard, i.e not
too high nor too low, and therefore depending on the magnification power of the lenses, it will range between 40X and
100oX.

Calculation of magnification = Magnification of objective lens/magnification of the eyepiece lens

The objective lens plays a vital role in not only enlarging the image but also making it clear for viewing, a feature known
as resolution. Resolution according to Prescott, is the ability of a lens to separate or distinguish between small objects
closely linked together.

Whereas the eyepiece magnifies the image at the end of the viewing, its magnification range is lower than that of the
objective lens at 8X-12X (10X standard) and that of the objective lens at 40X-100X, magnification, and resolution of the
microscope is highly dependant on the objective lens.

Applications of the Bright Field Light Microscope (Compound light microscope)

Vastly used in Microbiology, this microscope is used to view fixed and live specimens, that have been stained with basic
stains. This gives contrast for easy visibility under the microscope. Therefore it can be used to identify basic bacteria cells
and parasitic protozoans such as Paramecium.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Light Microscope Free Worksheet

Phase Contrast Microscope

• This is a type of optical microscope whereby small light deviations known as phase shifts occur during light
penetration into the unstained specimen. These phase shifts are converted into the image to mean, when light passes
through the opaque specimen, the phase shifts brighten the specimen forming an illuminated (bright) image in the
background.

• The phase-contrast microscope produces high contrast images when using a transparent specimen more so those of
microbial cultures, thin tissue fragments, cell tissues, and subcellular particles.

• The principle behind the working of the phase-contrast microscope is the use of an optical method to transform a
specimen into an amplitude image, that’s viewed by the eyepiece of the microscope.

• The PCM can be used to view unstained cells also known as the phase objects, which means that the morphology of
the cell is maintained and the cells can be observed in their natural state, in high contrast and efficient clarity. This is
because if the specimens are stained and fixed, they kill most cells, a characteristic that is uniquely undone by the
brightfield light microscope.

• The shifts that occur during light penetration, become converted to changes in amplitude which causes the image
contrast.

• Coupled with contrast-enhancing elements such as fluorescence, they produce better visuals of the specimens’
image.

Parts of the Phase Contrast Microscope

The instrumentation of the Phase Contrast Microscope is based on its light pathways from receiving the source of light to the
visualization of the image.

Therefore its sequentially made up of:

• Light source (Mercury arc lamp)

• Collective lens

• Aperture

• Condenser

• Condenser annular

• Specimen

• Objective

• Phase plate

• Deflected light

• Phase ring

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Phase Contrast Microscopy

The functioning of the Phase Contrast microscope

• The change is caused by the deviated scattered (Deflected) light and the undeviated light that reaches the specimen
which is absorbed, create at a certain wavelength, producing color. The difference created by the scattered light and
that of the absorbed light is known as amplitude variations. These amplitude variations are sensitive to allowing
visualization by photographic equipment like the Phase Contrast Microscope, hence seen by the human eye.

• The Condenser of the phase-contrast microscope has an opaque disk that is known as an annular ring, with a
transparent ring that produces a cone of light, that passes through a specimen. Due to light variations some light
bends at the specimen, caused by variations in light density, forming an image at the objective lens. The undeviated
light will strike the phase ring on the phase plate and the deviated light will miss the phase ring passing through the
phase plate directly, this forms an image.

The Phase-Contrast Microscope is designed with objective lenses that have the ability to perform multiple functions when
combined with contrast-enhancing techniques, for example, fluorescence. The objective lenses are located in the internal
phase plate with variation in the light absorption and phase displacement i.e undiffraction, creating a wide spectrum for
contrasting the specimen and forming a strong contrast in the background.

Applications of Phase-Contrast Microscope

• Determine morphologies of living cells such as plant and animal cells

• Studying microbial motility and structures of locomotion

• To detect certain microbial elements such as the bacterial endospores

Dark-Field Light Microscope

This is a specialized type of bright field light microscope that has several similarities to the Phase-Contrast Microscope. To
make a dark field Microscope, place a darkfield stop underneath and a condenser lens which produces a hollow cone beam of
light that enters the objective only, from the specimen (Prescott, pg 22).

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
This technique is used to visualize living unstained cells. This is affected by the way illumination is done on the specimen in
that, when a hollow cone beam of light is transmitted to the specimen, deviated light (unreflected/unrefracted) rays do not
pass through the objectives but the undeviated (reflected/refracted) light passes through the objectives to the specimen
forming an image.

This makes the surrounding field of the specimen appear black while the specimen will appear illuminated. This is enabled
by the dark background this the name, dark-field Microscopy.

Applications of the Dark Field Microscope

• It is used to visualize the internal organs of larger cells such as the eukaryotic cells

• Identification of bacterial cells with distinctive shapes such as Treponema pallidum, a causative agent of syphilis.

The Fluorescent Microscope

The above-discussed microscopes will normally produce images after a light has been transmitted and passed through the
specimen.

In the case of the fluorescent Microscope, the specimen emits light. How? By adding a dye molecule to the specimen. This
dye molecule will normally become excited when it absorbs light energy, hence it releases any trapped energy as light. The
light energy that is released by the excited molecule has a long wavelength compared to its radiating light. The dye molecule
is normally a fluorochrome, that fluoresces when exposed to the light of a certain specific wavelength. The image formed is
a fluorochrome-labeled image from the emitted light

The principle behind this working mechanism is that the fluorescent microscope will expose the specimen to ultra or violet
or blue light, which forms an image of the specimen that is emanated by the fluorescent light. They have a mercury vapor arc
lamp that produces an intense beam of light that passes through an exciter filter. The exciter filter functions to transmit a
specific wavelength to the fluorochrome stained specimen, producing the fluorochrome-labeled image, at the objective.

After the objective, there is a barrier filter that functions primarily to remove any ultraviolet radiation that may be harmful to
the viewer’s light, thus reducing the contrast of the image.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)
Fluorescence Microscopy

Applications of the Fluorescent Microscope

• Used in the visualization of bacterial agents such as Mycobacterium tuberculosis.

• Used to identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence

techniques by labeling the antibodies with fluorochromes.

• Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes

• It can also be used to differentiate between dead and live bacteria by the color they emit when treated with special
stains

Besides the above-discussed microscopes, there is one not commonly used microscope known as the Differential
Interference Contrast Microscopy. It is very similar to the phase-contrast microscope whereby the images are formed from
the variations in the light either deviated and or undeviated. The difference is, here two beams of light are emitted to the
specimen and focused by a prism. One beam passes through the prism to the specimen while another passes through the
glass slide clear area without the specimen. The two beams then combine and interfere with each other to form an image. It
can be used to view cell structures such as endospores, bacterial cell walls, nuclei, and granules for unstained specimens.

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SBT 111/ BOT 110/BOT 112/BIO 101 (Fundamentals of Botany)