This slide describes some methods that frequently use to study the structure of cells and cell organelles

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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