MICROSCOPY: IMAGING OF BIOLOGICAL SPECIMENS - A Guide to Methods in the Biomedical Sciences - page 90

is possible with phase contrast. DIC uses prisms to split light; when the
light recombines in a second prism, any differences in surface topology
will be reflected in an altered optical path, resulting in increased contrast.
The method produces monochromatic shadowed images where optical
path length gradients are introduced, which produces superior contrast
into the images.
C. Fluorescence and laser confocal
microarray microscopy
Introduction to standard (widefield)
fluorescence microscopy
Fluorescence microscopy takes advantage of fluorescently labeled
“probes” to visualize the location of a target molecule within cells or
tissues. Rather than visualizing the specimen itself, fluorescent micro-
scopes reveal the localization of a target molecule by illuminating the
fluorescent probe. These probes are usually antibodies (polyclonal or
monoclonal) or lectins (proteins that bind specific sugar residues), and
may be directly labeled with a fluor, or themselves specifically detected
with a secondary reagent that is labeled. Most fluorescent microscopes
use “epifluorescence”, meaning that the excitatory light is transmitted
through the objectives onto the specimen and not through the speci-
men. In this way, only the reflected excitatory light needs to be filtered,
which reduces “noise” in the signal.
Fluorescent molecules
Fluorescent molecules absorb light of a particular wavelength and
emit it at longer wavelengths. Fluorescent microscopes pass light
through “excitation” and “emission” filters that allow the visualization
of the fluorescent probe. A variety of probes that emit at different wave-
lengths are now available. This allows investigators to sequentially vi-
sualize a number of different target molecules within regions of the cell,
yielding a variety of colors including greens, reds, yellows, blues, etc.
This not only has greatly enhanced the ability of cell biologists to visu-
alize the localization and movement of proteins and organelles within
cells, but the pictures can have the most amazing “wow” effect! Fluo-
rescence microscopy is used to characterize the organization of cells,
and the compartments within cells (nucleus, Golgi, endoplasmic retic-
ulum, plasma membrane and endosomal compartments, etc.), as well
as the vesicles and tubules that transport “cargo” between these com-
partments. In order to label intracellular proteins with specific antibodies,