PART I
General Biochemical and
Biophysical Methods
Topics covered:
• Microscopy
• Spectrophotometry
• Fluorescence and Flow Cytometry
• Radioactivity
• pH and Buffers
• Centrifugation
8
, CHAPTER 1--MICROSCOPY
Cells are small and in almost all situations a microscope is needed to observe them and
their subcellular components. In fact the invention of the microscope led to the discovery and
description of cells by Hooke in 1655. The microscope is still an extremely important tool in
biological research. The light microscope has a limited capability in regards to the size of a
particle that can be examined. The electron microscope provides additional resolution that
allows for the examination of subcellular structures and even molecules.
LIGHT MICROSCOPY
The principal of light microsco-
py is to shine light through a specimen
and examine it under magnification.
The major optical parts of a microscope
are the objective lens, the eyepiece, the
condenser and the light source. The
objective lens functions to magnify the
object. The high degree of magnifica-
tion of the objective lens results in a
small focal length and the magnified
image actually appears directly behind
the objective. The eyepiece functions to
deliver this image to the eye or camera.
Eyepieces also magnify the image, but it
is an empty magnification. In other
words, the eyepiece enlarges the image Major components of a light microscope
but does not increase the ability to see fine details (i.e., the resolution). The condenser functions
to focus the light source on the specimen. The condensor also eliminates stray light and
provides an uniform illumination. An iris diaphragm which controls the amount of light
reaching the specimen is also associated with the condenser lens. In addition, the light intensity
can also be controlled by adjusting the voltage applied to the lamp on some microscopes.
Before using a microscope (Box) it is also important to check 1. Center light source
that all of the optical components are centered on an optical axis and all components
so that the best image and resolution are obtained. Aligning the on optic axis.
optical components is usually simple (see instructions manual 2. Focus objective.
for the particular microscope) and needs to be done periodically. 3. Focus condenser.
The specimen is then placed on the stage and the objective lens 4. Adjust illumination.
is focused. The quality of the image produced is highly
dependent on the illumination. The position of the condenser lens is adjusted so that the light is
focused on the specimen and the intensity of the illumination is adjusted. On better microscopes
the illumination can be controlled by both adjusting the diameter of the iris and by adjusting the
voltage applied to the lamp. The amount of illumination is important for controlling resolution
vs. the contrast and the depth of field (see Box for definitions). Resolution and contrast are
antagonistic in that improving one results in a loss of the other. Resolution is increased by
9
,increasing the amount of light. However, the brighter light leads to a loss in contrast. The user
must decide upon the optimal mix of contrast and resolution by adjusting both the voltage (i.e.,
intensity or brightness) of the lamp and the iris diaphragm. The iris diaphragm also has some
effect on the depth of field.
Resolution The ability to discern fine details. Typically expressed as a
linear dimension describing the smallest distance needed
between 2 objects so that both are seen.
Contrast Contrast refers to the number of shades in a specimen.
More shades decreases the contrast, but increases the
amount of information (also called dynamic range).
Depth of Field Refers to the thickness of the specimen that will be in
acceptable focus.
Sample Preparation
Specimens can be examined by simply placing them on a glass microscope slide under a
glass cover slip. However, it is usually necessary to prepare and stain the samples before
examination by microscopy. Fixation is a process by which cells are preserved and stabilized.
Common fixatives include: acids, organic solvents, formaldehyde and glutaraldehyde (see
Appendix for more discussion about aldehyde fixatives). These treatments affix macromole-
cules in position. For example, glutaraldehyde chemically cross-links the primary amines of
neighboring proteins and organic solvents precipitate proteins and other macromolecules.
Thick samples, such as tissues, will need to
cut into thin sections. Following fixation the sample
or cells are embedded into a supporting medium.
Paraffin is a common embedding medium for light
microscopy as well as various plastic resins.
Sectioning is carried out with a microtome (Figure).
The microtome cuts the specimen into thin slices, or
sections, of a specified thickness. It is also possible to
collect the successive slices, called serial sections, and
therefore ascertain the three dimensional aspects of
the tissue or specimen being examined.
The image generated by microscopy depends
upon different components in the sample interacting with and impeding the light waves
differentially. Biological samples are fairly homogeneous (i.e., carbon-based polymers) and do
not greatly impede light. Therefore, it is often necessary to stain cells with dyes to provide more
contrast. Different dyes have different affinities for different subcellular components. For
example, many dyes specifically interact with nucleic acids (i.e., DNA and RNA) and will
differentially stain the cytoplasm and nucleus. These stained subcellular components will
differentially absorb the light waves and result in less light reaching the eyes or camera, and
thus appears darker. Furthermore, since the dyes only absorb certain wavelengths of light, the
10
, various structures within the specimen will exhibit
different colors. (See chapter on Spectrophotometry for a • Dark Field
more extensive discussion of chromaphores and light • Phase Contrast
absorption.) • Differential Interference
Contrast (or Normarski)
• Confocal Scanning
Variations to bright field (transmission) microscopy • Fluorescence
• Image Enhancement
Many modifications of light microscopy that have
specialized applications have been developed (Box). In
dark-field microscopy the specimen is illuminated from the side and only scattered light enters
the objective lens which results in bright objects against dark background. This is accomplished
through the use of an annular aperture that will produce a hollow cone of light that does not
enter the objective lens (see Figure). Some of the light hitting objects within the specimen will
be diffracted into the objective lens (see Figure Inset). The images produced by dark-field
microscopy are low resolution and details cannot be seen. Dark-field microscopy is especially
useful for visualization of small particles such as bacteria.
Dark Field Microscopy Optics Phase Shift vs Diffraction
Both phase contrast microscopy and differential-interference-contrast allow objects
that differ slightly in refractive index or thickness to be distinguished within unstained or living
cells. Differences in the thickness or refractive index within the specimen result in a differential
retardation of light which shifts the phase or deviates the direction of the light (Figure). During
phase contrast microscopy the phase differences are converted to intensity differences by
special objectives and condensers. Normarski optics use special condensers and objectives to
recombine incident and diffracted light waves from a single source at the plane of the image. In
both methods the interference effects between the incident and diffracted light enhance small
differences in the refractive index or thickness of the specimen and leads to an increased
resolution without staining.
In fluorescence microscopy a fluorochrome is excited with ultraviolet light and the
resulting visible fluorescence is viewed. This produces a bright image in a dark background.
11
General Biochemical and
Biophysical Methods
Topics covered:
• Microscopy
• Spectrophotometry
• Fluorescence and Flow Cytometry
• Radioactivity
• pH and Buffers
• Centrifugation
8
, CHAPTER 1--MICROSCOPY
Cells are small and in almost all situations a microscope is needed to observe them and
their subcellular components. In fact the invention of the microscope led to the discovery and
description of cells by Hooke in 1655. The microscope is still an extremely important tool in
biological research. The light microscope has a limited capability in regards to the size of a
particle that can be examined. The electron microscope provides additional resolution that
allows for the examination of subcellular structures and even molecules.
LIGHT MICROSCOPY
The principal of light microsco-
py is to shine light through a specimen
and examine it under magnification.
The major optical parts of a microscope
are the objective lens, the eyepiece, the
condenser and the light source. The
objective lens functions to magnify the
object. The high degree of magnifica-
tion of the objective lens results in a
small focal length and the magnified
image actually appears directly behind
the objective. The eyepiece functions to
deliver this image to the eye or camera.
Eyepieces also magnify the image, but it
is an empty magnification. In other
words, the eyepiece enlarges the image Major components of a light microscope
but does not increase the ability to see fine details (i.e., the resolution). The condenser functions
to focus the light source on the specimen. The condensor also eliminates stray light and
provides an uniform illumination. An iris diaphragm which controls the amount of light
reaching the specimen is also associated with the condenser lens. In addition, the light intensity
can also be controlled by adjusting the voltage applied to the lamp on some microscopes.
Before using a microscope (Box) it is also important to check 1. Center light source
that all of the optical components are centered on an optical axis and all components
so that the best image and resolution are obtained. Aligning the on optic axis.
optical components is usually simple (see instructions manual 2. Focus objective.
for the particular microscope) and needs to be done periodically. 3. Focus condenser.
The specimen is then placed on the stage and the objective lens 4. Adjust illumination.
is focused. The quality of the image produced is highly
dependent on the illumination. The position of the condenser lens is adjusted so that the light is
focused on the specimen and the intensity of the illumination is adjusted. On better microscopes
the illumination can be controlled by both adjusting the diameter of the iris and by adjusting the
voltage applied to the lamp. The amount of illumination is important for controlling resolution
vs. the contrast and the depth of field (see Box for definitions). Resolution and contrast are
antagonistic in that improving one results in a loss of the other. Resolution is increased by
9
,increasing the amount of light. However, the brighter light leads to a loss in contrast. The user
must decide upon the optimal mix of contrast and resolution by adjusting both the voltage (i.e.,
intensity or brightness) of the lamp and the iris diaphragm. The iris diaphragm also has some
effect on the depth of field.
Resolution The ability to discern fine details. Typically expressed as a
linear dimension describing the smallest distance needed
between 2 objects so that both are seen.
Contrast Contrast refers to the number of shades in a specimen.
More shades decreases the contrast, but increases the
amount of information (also called dynamic range).
Depth of Field Refers to the thickness of the specimen that will be in
acceptable focus.
Sample Preparation
Specimens can be examined by simply placing them on a glass microscope slide under a
glass cover slip. However, it is usually necessary to prepare and stain the samples before
examination by microscopy. Fixation is a process by which cells are preserved and stabilized.
Common fixatives include: acids, organic solvents, formaldehyde and glutaraldehyde (see
Appendix for more discussion about aldehyde fixatives). These treatments affix macromole-
cules in position. For example, glutaraldehyde chemically cross-links the primary amines of
neighboring proteins and organic solvents precipitate proteins and other macromolecules.
Thick samples, such as tissues, will need to
cut into thin sections. Following fixation the sample
or cells are embedded into a supporting medium.
Paraffin is a common embedding medium for light
microscopy as well as various plastic resins.
Sectioning is carried out with a microtome (Figure).
The microtome cuts the specimen into thin slices, or
sections, of a specified thickness. It is also possible to
collect the successive slices, called serial sections, and
therefore ascertain the three dimensional aspects of
the tissue or specimen being examined.
The image generated by microscopy depends
upon different components in the sample interacting with and impeding the light waves
differentially. Biological samples are fairly homogeneous (i.e., carbon-based polymers) and do
not greatly impede light. Therefore, it is often necessary to stain cells with dyes to provide more
contrast. Different dyes have different affinities for different subcellular components. For
example, many dyes specifically interact with nucleic acids (i.e., DNA and RNA) and will
differentially stain the cytoplasm and nucleus. These stained subcellular components will
differentially absorb the light waves and result in less light reaching the eyes or camera, and
thus appears darker. Furthermore, since the dyes only absorb certain wavelengths of light, the
10
, various structures within the specimen will exhibit
different colors. (See chapter on Spectrophotometry for a • Dark Field
more extensive discussion of chromaphores and light • Phase Contrast
absorption.) • Differential Interference
Contrast (or Normarski)
• Confocal Scanning
Variations to bright field (transmission) microscopy • Fluorescence
• Image Enhancement
Many modifications of light microscopy that have
specialized applications have been developed (Box). In
dark-field microscopy the specimen is illuminated from the side and only scattered light enters
the objective lens which results in bright objects against dark background. This is accomplished
through the use of an annular aperture that will produce a hollow cone of light that does not
enter the objective lens (see Figure). Some of the light hitting objects within the specimen will
be diffracted into the objective lens (see Figure Inset). The images produced by dark-field
microscopy are low resolution and details cannot be seen. Dark-field microscopy is especially
useful for visualization of small particles such as bacteria.
Dark Field Microscopy Optics Phase Shift vs Diffraction
Both phase contrast microscopy and differential-interference-contrast allow objects
that differ slightly in refractive index or thickness to be distinguished within unstained or living
cells. Differences in the thickness or refractive index within the specimen result in a differential
retardation of light which shifts the phase or deviates the direction of the light (Figure). During
phase contrast microscopy the phase differences are converted to intensity differences by
special objectives and condensers. Normarski optics use special condensers and objectives to
recombine incident and diffracted light waves from a single source at the plane of the image. In
both methods the interference effects between the incident and diffracted light enhance small
differences in the refractive index or thickness of the specimen and leads to an increased
resolution without staining.
In fluorescence microscopy a fluorochrome is excited with ultraviolet light and the
resulting visible fluorescence is viewed. This produces a bright image in a dark background.
11
