Introduction to the
Study of Cell and
Molecular Biology (3)
Techniques and Methods of
Studying Cells
This third lecture on the introduction to the study of the Cell and Molecular Biology is
particularly on the Techniques and Methods of Studying Cells.
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Topic
Introduction (Overview)
A.The Cellular Basis of Life
B.Different Cell Types
C.Techniques and Methods of Studying Cells
This is the third and last part of the three part topic on the introduction to cell and
molecular biology.
I will enumerate some common techniques here, other will be mention as we go
through the different topics in this course.
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Microscopy
It is the technical field of using microscopes to
view samples or objects
Magnification is the process of enlarging an object
only in appearance.
Since our naked eyes are not capable of seeing cells, microscopy is the most basic
technique in cell biology. It is the technical field of using microscopes to view samples
or objects making smaller objects appear bigger than it is. This is where the term
magnification comes in. Magnification is the process of enlarging an object only in
appearance.
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Microscopy
Resolution is the ability to tell two
points apart as separate points.
Resolving power is the
microscope's ability to
produce a clear image.
Depth of Field (DOF) is the
distance between the closest
and farthest objects in an
image that appears
acceptably sharp.
Microscopy is not only enlarging an object but on producing a clear image. Clarity is
based on the resolution or is the ability to tell two points apart as separate points. A
microscope’s resolving power is its ability to produce a clear image.
Depth of Field (DOF) is the distance between the closest and farthest objects in an
image that appears acceptably sharp.
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1.4 Light Microscopy
- employs visible light to detect small objects
- is probably the most well-known and well-used research tool
in biology to magnify objects
- the challenge of viewing small objects lies not just in getting
enough magnification
- challenges are in:
• obtaining sufficient contrast
• finding the focal plane
• obtaining good resolution
• recognizing the subject when one sees it
Light microscopy employs visible light to detect small objects. It is
probably the most well-known and well-used research tool in biology to
magnify objects. The challenge of viewing small objects lies not just in
getting enough magnification but challenges are in obtaining sufficient
contrast, finding the focal plane, obtaining good resolution and
recognizing the subject when one sees it.
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1.4 Light Microscopy
Types of light Microscopy: BRIGHT-FIELD
➢ light from an incandescent source
is aimed toward a lens beneath the
stage (the condenser)
➢ through the specimen
➢ through an objective lens
➢ through a second magnifying lens
(the ocular or eyepiece)
➢ to the eye
• We see objects in the light path
because natural pigmentation or
stains absorb light differentially
• or because they are thick enough to
absorb a significant amount of light
despite being colorless
There are several type of light microscopy. The most common one is the bright-field
wherein light from an incandescent source is aimed toward a lens beneath the stage
(the condenser), through the specimen, through an objective lens, through a second
magnifying lens (the ocular or eyepiece) to the eye.
In bright field microscopy, we see objects in the light path because natural
pigmentation or stains absorb light differentially or because they are thick enough to
absorb a significant amount of light despite being colorless.
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1.4 Light Microscopy
Types of light Microscopy: BRIGHT-FIELD
With Iodine
Live and unstained
Stained with Malachite
Green
These are cells of Tetrahymena captured under bright-field microscope. The live
unstained cells are observed in its natural mobility, however, its internal structures
are more distinguished using dyes that absorbs light differently.
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1.4 Light Microscopy
Types of light Microscopy: PHASE-CONTRAST
• some regions appear brighter or darker
• is preferable to bright field microscopy when high magnifications (400x, 1000x) are
needed and the specimen is colorless or the details so fine that color does not
show up well.
• Cilia and flagella, for example, are nearly invisible in bright field but show up in
sharp contrast in phase contrast.
• Amoebae look like vague outlines in bright field, but show a great deal of detail in
phase. Most living microscopic organisms are much more obvious in phase
contrast.
Bright-field Phase-contrast
Phase- contrast microscopy is another types of light Microscopy wherein some
regions appear brighter or darker. It is preferable to bright field microscopy when high
magnifications (400x, 1000x) are needed and the specimen is colorless or the details
so fine that color does not show up well.
Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp
contrast in phase contrast.
Amoebae look like vague outlines in bright field, but show a great deal of detail in
phase-contrast. Most living microscopic organisms are much more obvious in phase-
contrast.
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1.4 Light Microscopy
Types of light Microscopy: NOMARSKI DIFFERENTIAL-INTERFERENCE-
CONTRAST MICROSCOPY
• The thickness of most specimens prevents all parts from coming into focus all
at once, limiting the usefulness of higher magnification lenses.
• D.I.C. and related optics give a specimen a three dimensional appearance
that is not unlike the appearance of a specimen in a scanning electron
microscope.
• These methods enhance depth of focus so that thicker specimens can be
observed at higher magnifications.
Bright-field Normanski DIC
The thickness of most specimens prevents all parts from coming into focus all at
once, limiting the usefulness of higher magnification lenses. Nomarski differential
interference-contrast microscopy or D.I.C. give a specimen a three dimensional
appearance that is not unlike the appearance of a specimen in a scanning electron
microscope. These methods enhance depth of focus so that thicker specimens can be
observed at higher magnifications.
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1.4 Light Microscopy
Types of light Microscopy: DARK-FIELD
• excludes the unscattered beam from the image
• As a result, the field around the specimen (i.e., where there is no
specimen to scatter the beam) is generally dark.
Bright-field Dark-field
Dark-field light microscopy excludes the unscattered beam from the image. As a
result, the field around the specimen (i.e., where there is no specimen to scatter the
beam) is generally dark.
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1.5 Electron Microscopy
Electron microscope
uses a particle
beam of electrons to
illuminate a specimen
2 Types:
• Transmission (TEM)
• Scanning (SEM)
The second major type of microscopy is the Electron microscopy which uses a particle
beam of electrons to illuminate a specimen.
2 Types of electron microscope are: Transmission (TEM) and Scanning (SEM).
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1.5 Electron Microscopy
Types of Electron Microscopy:
Scanning Electron
• External views
• 3-D appearance
Transmission Electron
• Views cross-sections of
images
Inside a cell
• 2-D views
SEM provides external views and produce 3-D appearance while TEM
views cross-sections of images Inside a cell and produce 2-D views.
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1.5 The Fractionation and
analysis for cell’s contents
Differential Centrifugation
- used to separate organelles
and other sub-cellular
particles on the basis of
sedimentation rate
- The preparative
ultracentrifuge
Now lets mention some examples on methods for the fractionation and
analysis for cell’s contents.
One is differential centrifugation which is used to separate organelles
and other sub-cellular particles on the basis of sedimentation rate. Thus,
the first step is the preparative ultracentrifuge.
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1.5 The Fractionation and
analysis for cell’s contents
Differential Centrifugation
• Repeated centrifugation at progressively
higher speeds will fractionate homogenates
of cells into their components.
• In general, the smaller the subcellular
component, the greater is the centrifugal
force required to sediment it.
Repeated centrifugation at progressively higher speeds will fractionate homogenates
of cells into their components.
In general, the smaller the subcellular component, the greater is the centrifugal force
required to sediment it as shown in the right figure.
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1.5 The Fractionation and
analysis for cell’s contents
Paper Chromatography
• After the sample has been applied to one end of the paper (the "origin") and
dried, a solution containing a mixture of two or more solvents is allowed to flow
slowly through the paper by capillary action.
• Different components in the sample move at different rates in the paper
according to their relative solubility in the solvent that is preferentially adsorbed
onto the fibers of the paper.
Paper Chromatography is another method of fractionation.
After the sample has been applied to one end of the paper (the "origin") and dried, a
solution containing a mixture of two or more solvents is allowed to flow slowly
through the paper by capillary action.
Different components in the sample move at different rates in the paper according to
their relative solubility in the solvent that is preferentially adsorbed onto the fibers of
the paper.
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1.5 The Fractionation and
analysis for cell’s contents
Column Chromatography
• The sample is applied to the top of a
cylindrical glass or plastic column filled with a
permeable solid matrix, such as cellulose,
immersed in solvent.
• Then a large amount of solvent is pumped
slowly through the column and is collected in
separate tubes as it emerges from the bottom.
• Various components of the sample travel at
different rates through the column and are
thereby fractionated into different tubes.
HPLC and GC
In column chromatography, the sample is applied to the top of a cylindrical glass or
plastic column filled with a permeable solid matrix, such as cellulose, immersed in
solvent. Then a large amount of solvent is pumped slowly through the column and is
collected in separate tubes as it emerges from the bottom.
Various components of the sample travel at different rates through the column and
are thereby fractionated into different tubes.
Analytical chromatographic methods includes HPLC or High-performance liquid
chromatography and gas chromatography. Its your task to research the principles
behind these other chromatographic techniques.
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1.5 The Fractionation and
analysis for cell’s contents
Gel Electrophoresis
• separation and analysis of
macromolecules
(DNA, RNA and proteins) and their
fragments, based on their size and
charge.
Gel electrophoresis is used to separate and analyze of macromolecules (such
DNA, RNA and proteins) and their fragments, based on their size and charge.
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1.5 The Fractionation and
analysis for cell’s contents
Gel Electrophoresis
Agarose gel electrophoresis:
1) DNA is extracted
2) Isolation and amplification of DNA
3) DNA added to the gel wells
4) Electric current applied to the gel
5) DNA bands are separated by size
6) DNA bands are stained.
These are the basic steps in agarose gel electrophoresis: first, DNA (or other
macromolecule) is extracted; then followed by the Isolation and amplification of
DNA. Next, DNA added to the gel wells the electric current applied to the gel. After
some time, DNA bands are separated by size. Lastly, DNA bands are stained.
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1.5 The Fractionation and
analysis for cell’s contents
Western Blotting or immunoblotting
• technique by which an individual
protein is visualized amid thousands of
other proteins in a given sample.
• The technique uses sodium dodecyl
sulfate (SDS)-polyacrylamide gel
electrophoresis (SDS-PAGE) to
separate thousands of proteins
present in a sample.
Western Blotting or immunoblotting is a technique by which an individual protein is
visualized amid thousands of other proteins in a given sample.
The technique uses sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(SDS-PAGE) to separate thousands of proteins present in a sample.
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1.5 Other Techniques
Protein Structure
➢X-ray crystallography
➢NMR spectroscopy
RT-PCR
Monoclonal Antibodies
Gene Knockout Mice
There are several other techniques that are not discussed here. Methods in studying
cells is a dynamic field in cell biology and is evolving as the need arise.
You may also read about X-ray crystallography and NMR spectroscopy to study
Protein Structure. Also I suggest you research on RT-PCR, used for corona virus
detection; also on monoclonal antibodies and applications of Gene Knockout Mice.
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