Contribution of microscope to the expansion of knowledge on cells and

cellular organization
Microscopes
Advancement of the cytology is mostly based on the microscopy. The discovery and early
study of cells progressed with the invention of microscope. Microscope is the instrument that
use to observe magnified images of cell and intracellular components. There are two main types
microscopes;
1. Light microscope
2. Electron microscope
Two important parameters found in a microscope are;
1. Magnification
2. Resolution

Magnification is the ratio of an object’s image size to its actual size. Usually the maximum
magnification of compound light microscope is 1000 times the actual size of the specimen.

Resolution power is minimum distance between two points that can be distinguished as
separate points
Ex: Resolution power of human eye is 0.1mm
Resolution power of compound light microscope is 0.2μm/ 200 nm
Resolution power of transmission electron microscope is 0.2 nm
Resolution power of scanning electron microscope is 5 – 20 nm

Resolution power is inversely proportional to the wavelength of light or electron beam used in
microscope. It is a measure of the clarity of the image. Magnification is limited due to the
resolution.

Light microscope
In light microscope, visible light is passed through the specimen and then through glass lenses.
Convex glass lenses are used in light microscopes. These lenses refract the light in such a way
that the image of the specimen is magnified as it is projected into the eye. Based on the number
of lenses used, there two basic types of light microscopes.
They are;

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 1. Simple light microscope (Magnifying lens) - This is a single convex lens.
2. Compound light microscope – Two convex lenses as Objective lens and eye piece are
used.

The compound light microscope

Compound light microscopes are commonly used in school laboratories and it is used in
medical laboratories as a diagnostic tool.

Practical No 2: Identification of parts and functions of the light microscope and use of
light microscope to observe specimens

a) Parts of compound light microscope

Eye piece lens

Ocular tube

Coarse adjustment

Fine adjustment

Arm Nose piece

Objective lenses
Stage

Stage clips

Diaphragm lever

Condenser

Mirror / light
source can be used

Base

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Functions of main parts of compound light microscope
1. Mirror – reflect light and supply light towards the object/specimen on slide
2. Condenser – This is glass convex lens. It converges light and focuses light on the object
3. Diaphragm – This has an aperture in the middle. Light converged by condenser moves
towards the specimen through diaphragm. The size of aperture can be controlled using
diaphragm lever. Therefore, diaphragm controls the amount pf light moves to the specimen.
4. Coarse adjustment knob – Use for rough focusing of the specimen. By rotating this,
distance between specimen and the objective lens can be changed to a greater extent.
5. Fine adjustment knob - Use for precise focusing of the specimen. By rotating this, distance
between specimen and the objective lens can be changed slightly.
6. Objective lens:
There are three common objective lenses.
1) High power objective lens – Magnification is ×40
2) Mid power objective lens - Magnification is ×10
3) Low power objective lens - Magnification is ×04
Light from an object (specimen on the slide) passes first through objective lens. Then produce
a magnified image within the ocular tube. This image forms within ocular tube then acts as an
object for the second lens (the eye piece lens) which further magnifies it and forms a magnified
image within eye.
7. Eye piece lens – magnifies the image that form within the ocular tube. Magnification of
eye piece can be ×10/×15/×20.
The total magnification is hence the product of the magnification of each lens.
e.g- .If magnification of Objective lens = ×40, eyepiece =×15
Total magnification is =15 × 40= ×600 time magnified

b) Preparation of temporary mount of onion/ Rhoeo epidermal peels.

Steps of preparation of a temporary mount:
• Make thin epidermal peels of onion /Rhoeo (Tradescantia) and place in water in a watch
glass or Petri dish.
• Transfer a piece of onion Rhoeo (Tradescantia) epidermal peel into a drop of water on the
center of a clean glass slide using a fine paint brush.
• Hold the cover slip at the edge of the drop of water, with the help of a mounting needle,
and gently lower the cover slip while supporting it with the needle onto the drop of water.
• Cover the specimen/ tissue sample by cover slip without trapping air bubbles under the
cover slip.

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c) Observation of specimens through compound light microscope
• Place the slide on the stage of the microscope, clip it and observe under low power objective
lens.
• Looking through the eye piece, move the slide to bring the object into position for study.
• By observing through eye piece, adjust the mirror and condenser to give optimum
illumination to the object for clear viewing. (a clear visual field should be observed)
• Use the coarse focusing knob to get the image as clear as possible.
• Use the fine focusing knob to improve the quality of the image. (for fine focusing to get
more sharper image)
• Study and note the structures visible.
• Rotate the nose piece and bring the medium power objective lens into position.
• Adjust the focusing to get a sharp image.
If further magnified image should be observed;
• Bring the high power into position
• Use the fine focusing knob only to make the image sharp.
• Study and record what you observe under low, medium and high power.
Note: Under high power only fine adjustment knob should be used for focusing. If coarse
adjustment knob is used, both the lens and the specimen can be damaged.
Follow the steps given above to observe a drop of water from paddy field, hay infusion, pond
water and cells obtained from buccal cavity lining

Oil immersion microscopy

In oil microscopy, objective lens with magnification power of ×100 is used. A specialized oil
is placed in between the objective lens and the specimen as shown in the above diagram. In

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normal light microscopy light rays move to objective lens through air after passing the
specimen. (See the diagram (a)). But in oil immersion microscopy light moves through oil.
This provides more sharper and clear image, because oil has high refraction index than air.
Then light converges better and moves towards lens.

The Electron Microscope

The resolution power of the light microscope becomes limited by the comparatively high
wavelength of light. The resolution power is inversely proportional to the wavelength.
Due to this, scientists considered the use of other forms of radiations with comparatively shorter
wavelengths.
As a result, electron microscopes were developed. In electron microscopy, a beam of electrons
is focused through the specimen or on to its surface. Glass lenses can’t be used in electron
microscopes. Therefore, electro-magnets are used to focus the electron beam and to get
magnified image. In electron microscopes dehydrated dead specimens are placed inside a
vacuum. Living specimens can’t be observed through electron microscopes.

Electron beam has very low wavelength compared to light. Therefore, resolution power of
electron microscope becomes higher than light microscope. Due to high resolution,
magnification is also high in electron microscope. This means, that in theory, the electron
microscope should be able to magnify objects up to 1×108 times. In practice, it magnifies just
over 5×105 times.
Therefore, electron microscopes have revealed many organelles and other sub cellular
structures those were impossible to resolve with the light microscopes.
There are two types of electron microscopes.
1. Transmission electron microscopes (TEM)
2. Scanning electron microscopes (SEM)

Transmission electron microscopes

It is used to study the internal ultra/ fine structures of cells. In this microscope, a beam of
electrons is passed through a thin, especially prepared slice of material. A very thin specimen
is used, which allows penetrating the electron beam through the specimen.
Specimens are stained with heavy metals which attach more to certain cellular structures than
other areas. These heavy metals reflect electrons passing through the specimen. Since heavy
metals are not uniformly attached to all intracellular structures, electrons are reflected by these

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structures in a different pattern and form an image. Image reflects the pattern of electrons
passed through the specimen, which displays on a screen. While electrons pass through the
specimen, more electrons may get displayed in regions where structures were densely stained.
Three dimensional images can’t be observed through TEM, only two-dimensional images can
be observed. Resolution power and magnification of TEM are comparatively higher than SEM.

Electron micrograph
Transmission electron microscopes

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Scanning electron microscopes
This instrument is ideal to observe the surface view of objects in three dimensional
appearances. In this instrument, a fine beam of electrons is reflected from the surface of
specimen and form images. Electrons do not penetrate the specimen. Therefore, comparatively
thick specimens can be used in SEM than TEM.
Specimen is mostly coated with gold prior to observation. Here the specimen scatters many
electrons whereas others are absorbed. Three dimensional images can be observed using SEM.

Scanning electron microscope

Surface view of pollen grains as observed through scanning electron microscope

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Differences between compound light and electron microscope

Compound light microscope Electron microscope

Glass lenses are used to focus the light rays Powerful electro-magnets are used to focus
beam of electrons
Image is directly detected by naked eye Not directly detected by naked eye.
Micrographs are used or image is projected
to a screen
Living and nonliving objects can be Only non- living objects are observed
observed
Actual colour of the object can be observed. Actual colour cannot be observed. Only
black and white images can be observed.
Images are developed.
Dyes used to stain the object Heavy metals are used to stain the object.

Specimens are mounted on glass slides Specimens are mounted on Cu grid

Specimen is exposed to air Specimen is placed inside a vacum

Has comparatively low resolution power Has comparatively higher resolution power
and magnification and magnification

Historical background of the cell and analyses the structure and functions of
the sub cellular units
Cell theory
All organisms are composed of cells.
Recall the hierarchy of life, the levels of organization mentioned earlier. The basic unit which
can be called “living” is the cell, which may form a single celled organism
(e.g.Chlamydomonas, Yeast) or a multi-cellular plant or animal. The cell is the basic structural
and functional unit of life.
The level of organization of matter represented by a cell shows all the characteristics of life.
Any stage below level of a cell cannot be considered living.

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Main discoveries involved in proposing cell theory:
• Robert Hooke (1665) examined a cork using simple microscope and gave the term “CELL”
to describe the basic units.
• Anton Van Leeuwenhook (1650), a contemporary of Robert Hooke, was the first to
describe and record living single celled organisms, Euglena & bacteria
• Matthias Schleiden (1831), a botanist, studying plant tissues concluded that all plants
are made up of cells.
• Theodore Schwann a zoologist (1839) concluded that animal tissues are also made up of
cells.
• Rudolf Virchow (1855) showed that all cells arise from pre-existing cells by cell division,
Schleiden, Schwann and Virchow presented the ‘Cell Theory’ which included the following.
1.All organisms are composed of one or more cells.
2.The basic structural and functional unit of organisms is the cell.
3.All cells arise from pre-existing cells.

Organization of cells
There are two kinds of cellular organization
1. Prokaryotic cellular organization
Ex: Bacteria, Archaea bacteria and Cyanobacteria
2. Eukaryotic cellular organization
Ex: Protists, Fungi, Plants and Animals

All cells share certain basic features. They are;

• All cells are bounded by a plasma membrane which is a selective barrier
• Within the cell have, a semifluid, jelly like substance which is called cytosol. Subcellular
components are suspended within the cytosol.
• They carry DNA as genetic materials.
• Ribosomes are found in all cells

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 Eukaryotic cellular organization
Diagram of an Electron micrograph of an animal cell

Electron microscopic diagram of a plant cell

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Diagram of an electron micrograph of a plant cell

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Differences between animal cell and plant cell
Animal cell
1. Lack a cell wall, outer most layer is cell
membrane
2. Lack chloroplast and other plastids
3. Lack a large central vacuole
4. Lack glyoxisomes
5. Has centrioles
6. Has cell junctions like tight junctions,
anchor junctions and gap junctions
7. Has lysosomes
8. Cell membrane can produce endocytotic
and exocytotic vesicle
9. Some cells have microvilli
Plant cell
Has a cell wall as the outer covering
Has chloroplast and other plastids
Has a large central vacuole
Has glyoxisomes
Lack centrioles
Has plasmodesmata as the cell junction
Lysosomes are not abundant
Doesn’t form endocytotic and exocytotic
vesicles
Lack microvilli

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