MICROSCOPY: IMAGING OF BIOLOGICAL SPECIMENS - A Guide to Methods in the Biomedical Sciences - page 92

Figure 9. Comparison of immunofluorescence and laser confocal microscopy

can be controlled, but generally sections of are collected. These
sections are sufficient to resolve organelles at relatively high resolution,
but are too thick for details on vesicular compartments within the cell.
The design of the confocal microscope evolved over a period of 8 to
10 years to the type of system used today (reviewed in 36). The func-
tionality of confocal microscopy is dependent on computer software for
collecting images from the microscope. The key advantage of confocal
microscopy is that filtering is used to eliminate out-of-focus signals that
otherwise degrades the quality of the image created in standard wide-
field fluorescent microscopy. Figure 9 shows two fluorescent images of
B lymphocytes, one collected using standard immunofluorescence, the
other using laser confocal microscopy. Note the clarity of the confocal
image, while the image that is collected using a standard fluorescence
microscope is hazy due to the out of focus fluorescence. Only one probe
is used for these images: image the difference in clarity when 3 or 4 dif-
ferent fluorescent probes are combined to localize different proteins and
compartments within a single cell!
Because optical sections can be collected, a major advantage of con-
focal microscopy is that serial optical sections (in the X-Y plane) can be
collected. As a result, confocal microscopy not only provides informa-
tion on structures in an individual plane, but can collect multiple sections
in the z direction, generating a z-stack. Computer programs can then
reconstruct an optical image of the cell in 3-dimensions.
There are disadvantages of laser confocal microscopy. First, while
the optical sectioning rejects out-of-focus light, the laser generates
fluorescence even in these out-of-focus planes. This results in pho-
tobleaching of the non-imaged areas of the cells and, consequently,
significantly brighter images are required for confocal microscopy than
standard immunofluorescence. As a result, confocal microscopy is less