Chapter two discusses methods for studying cells, focusing on microscopy techniques that enhance the visibility of cellular structures. It covers light and electron microscopes, including their types, magnification capabilities, and resolution details, along with cell fractionation methods for isolating organelles. The chapter aims to provide students with foundational knowledge in microscopy to assist in their understanding of cellular biology.

1. Chapter Two
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Methods in the Study of Cells

2. Chapter objectives
At the end of this chapter the students will know:
 Microscopy and Types of Microscopy
 Magnification and Resolution
 Separation of cellular organelles
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3. Methods in the study of cell
Microscopy
Most cells are invisible to the human eye.
The smallest object a person can typically discern is about 0.2 mm (200 μm) in size.
Microscopes are instruments used to improve resolution so that cells and their internal structures can be seen.
Microscope (A Greek word, mikrós, "small" and skopeîn, "to look" or "see") is an instrument used to see
objects that are too small for the naked eye.
There are two basic types of microscopes:
 Light microscopes and
 Electron microscopes
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4. Light Microscope
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 Uses glass lenses and visible light to form a
magnified image of an object
 It has a resolving power of about 0.2 μm,
which is 1,000 times that of the human eye
 It allows visualization of cell sizes and shapes
and some internal cell structures

5. Light microscope- uses light and lenses to enlarge tiny objects
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6. Magnified Pollen grains – using light microscope
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7. Parts of Light Microscope
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 Body tube: The body tube is located between
the eye piece and nose piece. It is 160 mm in
length .

8. Parts of Microscope
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Ocular lens:
 These are also called as eye pieces located at
the top of the body tube.
 The ocular magnifies the image formed by
the objective lens.

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 Objective lens: These are mounted on the
revolving nose piece.
 The objective collects light rays from the object
and forms a magnified real image at some
distance within the body tube.
Parts of Microscope

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 Lens system: There are three types of lenses adapted
in light microscope ie., Condenser, Objective and
Ocular.
 Condenser lens: it is located below the stage and
above the mirror or light source.
 It collects and focuses the light rays into the plane of
the object.
 It does not take part in image formation.
 It just increases the intensity of light.
Parts of Microscope

11. Types of Light Microscope
 There are three types of Light Microscopes. These are:
1. Bright Field
2. Dark Field and
3. Phase Contrast
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12. Bright field microscopes
 A microscope that allows light rays to pass directly through
to the eye without being deflected by an intervening opaque
plate in the condenser.
 The bright-field light microscope is an instrument that
magnifies images using two lens systems.
 This is the conventional type of instrument encountered by
students in beginning courses in biology.
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13. Dark field microscope
Delicate transparent living organisms can be more easily observed with darkfield
microscopy than with conventional bright field microscopy.
This effect may be produced by placing a darkfield stop below the regular condenser.
Another application of darkfield microscopy is in the fluorescence microscope.
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14. Fluorescence Microscope
 Locations of specific molecules are revealed by labeling the molecules with fluorescent dyes or antibodies,
which absorb ultraviolet radiation and emit visible light.
 In this fluorescently labeled uterine cell, DNA is blue, organelles called mitochondria are orange, and
part of the cell’s “skeleton” (called the cytoskeleton) is green.
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15. Phase-Contrast Microscopy
A microscope that is able to differentiate transparent protoplasmic structures without staining and
killing.
It is the instrument of choice for studying living protozoans and other types of transparent cells.
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16. Electron microscope (EM)
 These are the most advanced type of microscope used in modern science.
 The electron microscope are powered by a beam of electron that strikes any to magnify
objects.
 Electron microscopes are used to studying cells and smaller particle of matter, as well as
large objects.
Light source
 The light source in this is electron gun and is located at the top of the microscope body.
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17. Electron microscope (EM)
It uses magnets to focus an electron beam
The resolving power of electron microscopes is about 0.5 nm, which is 400,000 times that
of the human eye.
This resolving power permits the details of many subcellular structures to be distinguished.
There are two main types of electron microscopes:
1. Scanning electron microscope (SEM)
2. Transmission electron microscope (TEM)
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18.  Electron microscope - electromagnetic lenses are used to
direct electrons onto the tiny specimen
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Electron microscope (EM)

19. Scanning electron microscope (SEM)
The SEM passes a beam of electrons over the specimen’s surface.
SEMs provide three-dimensional images of the specimen’s surface
SEMs can magnify objects up to 100,000 times.
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20. Pollen grains – scanning electron
microscope 3D
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Scanning electron microscope (SEM)

21. Transmission electron microscope (TEM)
The TEM transmits a beam of electrons through a very thinly
sliced specimen.
Magnetic lenses enlarge the image and focus it on a screen or
photographic plate.
Transmission electron microscopes can magnify objects up to
200,000 times.
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22. Magnification
On an image of a specimen it is useful to show how much larger/smaller the image is than the real
specimen. This is called magnification.
Magnification is the number of times larger an image is, than the real size of the object.
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23. How do we find the overall magnification of a light microscope?
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Eyepiece
Magnification
Objective
Magnification
Overall
Magnification
X10 X4 40
X10 X10 100
X10 X40 400
X10 X100 1000
Eyepiece
Objective lens

24. Con’t
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25. How to calculate Magnification?
To calculate magnification
 Using a ruler measure the size of a large clear feature on the image
 Measure the same length on the specimen
 Convert to the same units of measurement
Magnification = length on the image /length on the specimen
Length of the actual specimen = length on the image/Magnification
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26. Con’t
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 In this example the image of a Rose leaf the
magnification is X 0.83 and picture is 4.2cm
 This tells us the image is smaller than the real
specimen.
 The length of the real specimen = picture
length/Magnification 0.83 or 4.2cm/0.83 = 5.06
cm

27. Con’t
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28. Con’t
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31. Home Activity
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1. A student views an image of a cell magnified 250 times. The image is 150mm long. What is the actual
length of the sample in the image?
2. A sperm cell has a tail 50micro meter long. A student draws it 75mm long. How many times can be
magnified based the given data?

32. Solution
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33. Scale Bars
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 A scale bar is a line added to a drawing, diagram or
photograph to show the actual size of the structures.
 The scale bar in the picture allows you quickly to
determine the approximate size of a feature.
 The main feature in the micrograph is a nucleus with a
dark region called the nucleolus.
 Using the picture estimate the size of the nucleus and
its nucleolus.

34. Resolution
Resolution can be defined as the ability to distinguish between two separate points.
If the two points cannot be resolved, they will be seen as one point.
In practice, resolution is the amount of detail that can be seen – the greater the resolution, the greater the
detail.
The maximum resolution of a light microscope is 200 nm.
This means that if two points or objects are closer together than 200 nm they cannot be distinguished as
separate.
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35. The difference between magnification and resolution.
Magnification is the degree to which the size of an image is larger than the object itself.
Resolution is the degree to which it is possible to distinguish between two objects that are
very close together.
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36. Separation of cellular organelles
Each organelle has characteristics (size, shape and density for example) which make it different from other
organelles within the same cell.
If the cell is broken open in a gentle manner, each of its organelles can be subsequently isolated.
The process of breaking open cells is homogenization and the subsequent isolation of organelles is
fractionation.
Used to isolate different organelles of a Cell
 This enables individual organelle structures and functions to be studied
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37. Con’t
Isolating the organelles or cell fractionation requires the use of physical chemistry
techniques
Those techniques can range from the use of:
simple sieves
gravity sedimentation or
differential precipitation
ultracentrifugation of fluorescent labeled organelles in computer generated density
gradients.
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38. Steps of Cell Fractionation
There are three major steps of cell fractionation. These are:
1. Homogenization
2. Filtration
3. Ultra Centrifugation/ Fractionation
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39. 1. Homogenization
The first step in the preparation of isolated organelles is to obtain a "pure" sample for
further analysis
Cells which are part of a more solid tissue (such as liver or kidney) will first need to be
separated from all connections with other cells
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40. Con’t
Cells are broken open to release the contents
and organelles are then separated
The cells must be prepared in a cold isotonic
and buffered solution
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41. Con’t
 Cold: To reduce enzyme activity. When the cell breaks open enzymes are released which
could be damage the organelles
 Isotonic: Must be the same water potential to prevent osmosis as this could cause the
organelles to shrivel or burst.
 Buffered: the Solution has a PH buffer to prevent damage to organelles
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42. Con’t
 Homogenization techniques can be divided into those brought about by:
 Osmotic alterations
 Mortars, Pestles
 Blenders
 Compression/Expansion ….etc
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43. 2. Filtration
Homogenate will be Passed through a filter to remove unbroken tissue and cells
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44.  The most widely used technique for fractionating cellular
components is the use of centrifugal force
The Filtered Solution is spun at different speeds to in a
Centrifuge.
Material initially uniformly distributed in the solution
During spin, particles move with varying velocities down tube
Then organelles can be separated accordingly their size density.
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3. Fractionation/ Centrifugation

45. Con’t
 The centrifuge spins and the centrifugal force causes pellets of the most dense organelles to move to bottom.
Supernatant = liquid + most slowly regimenting component
pellet contains larger to smaller particles (usually mixture)
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46. Con’t
 The centrifuge is first spun at a low speed and the process is repeated at increasingly faster speeds
 Each time the supernatants (liquid) is removed, leaving behind a pallet of organelles
 The supernatant is then spun again to remove the next pellet of organelles
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47. Differential Centrifugation
Order of Pellet formation/ differential
of organelles
1. Nuclei--- (heaviest)
2. Chloroplasts (if it is plant cell)
3. Mitochondria
4. Lysosomes
5. Endoplasmic reticulum
6. Ribosomes ---- (lightest)
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48. Gravity Sedimentation
This can be accomplished by the simple use of gravity sedimentation
The samples are allowed to sit, and separation occurs due to the natural differences in
size and shape (density) of the cells
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