Biomedical Engineering Reference
In-Depth Information
the cells must first be fixed and permeabilized so that the antibodies can
gain entry to the cell. Common fixation agents include paraformaldehyde
and formaldehyde. Permeabilization of aldehyde-fixed cells is usually
accomplished with a detergent, such as Triton X-100 or saponin at very
low concentrations, lower than would normally be used to solubilize cells.
The agents that are used for fixation and permeabilization are strictly cell
and antibody dependent. Methanol can be used as both a fixative, which
also permeabilizes the cell. Finally, in some (rare) cases, permeabiliza-
tion without fixation can be accomplished with pore-forming agents such
as streptolysin-O (SLO). SLO is a toxin that comes from Streptococ-
cus aureus. It binds cholesterol in membranes and aggregates, forming
pores. SLO is used more often as a means to deplete cells of cytosolic
contents in systems that strive to define the cytosolic requirements for
intracellular vesicular trafficking by reconstitution.
Fixation and permeabilization of cells, also kills them cell, so that the
localization of target molecules are frozen in time. However, the local-
ization and movement of molecules can be visualized using chimeric
proteins, expressing variants of the green fluorescent protein (GFP) or
its spectral variants, such as YFP and CFP. Indeed, YFP and CFP are
the most common tags used for FRET analysis (see below).
Fluorescent microscopy can be combined with other methods that
are used to enhance contrast in cells, such as phase contrast and DIC.
These methods can be used to first localize the specimen on the slide,
since the illuminating light does not photobleach the probe, which de-
grades the sensitivity of the assay. In addition, contrast imaging and
fluorescent images can be superimposed in a single image to reveal
both the entire cell or tissue and the fluorescent probes.
There are limitations to standard fluorescence microscopy. The first
is the “out of focus spreading” that occurs when the entire specimen is
illuminated, even though the microscope is focused only on one plane
of the cell. This gives significant background and can frequently im-
pair resolution of internal structures. Second, the probes in the out of
focus areas, because they are illuminated, photobleach, resulting in
degradation of the intensity of staining. To overcome these problems,
other methods have been developed to visualize fluorescent probes
in individual planes of biological samples with high sensitivity and
resolution.
Laser confocal microscopy
Confocal microscopy is an optical sectioning method that selectively
collects images from a selected focal plane using a directed beam of light
from a laser. In a confocal microscope, the depth of the optical sections
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