Page 194 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
11

FLUORESCENCE MICROSCOPY

OVERVIEW

With light microscope optics adjusted for fluorescence microscopy, it is possible to
examine the distribution of a single molecular species in a specimen, and under special-
ized conditions, even detect individual fluorescent molecules. In contrast to other forms
of light microscopy based on object-dependent properties of light absorption, optical
path differences, phase gradients, and birefringence, fluorescence microscopy allows
visualization of specific molecules that fluoresce in the presence of excitatory light.
Thus, the amount, intracellular location, and movement of macromolecules, small
metabolites, and ions can be studied using this technique. Figure 11-1 shows one such
example: the distribution of an enzyme, -1,4-galactosyltransferase, in a tissue culture
cell. Typically, nonfluorescent molecules are tagged with a fluorescent dye or fluo-
rochrome, in order to make them visible. Examples of these are DAPI and the Hoechst
dyes used to directly label nuclear DNA or rhodamine-labeled phalloidin used to indi-
rectly label cytoplasmic actin filaments. Alternatively, fluorochrome-labeled antibodies
can be used to label fixed, permeabilized cells in a method known as immunofluores-
cence microscopy, the technique used in Figure 11-1. These techniques are commonly
used to visualize the distribution of certain proteins in a cell or to make visible specific
organelles, filaments, and biochemically distinct membrane regions. A variety of new
tagging methods is also employed, including inserting short DNA sequences of known
epitopes into the coding sequences of proteins (epitope tagging), constructing protein
chimeras with green or red fluorescent proteins (GFP, RFP), and several other methods.
Fluorescence microscopy has gained in popularity ever since Coons (1941) devel-
oped methods to conjugate proteins to fluorochromes, and as improvements in optics,
thin film technology, and opto-electronics increased the specificity and sensitivity of
detection of emitted fluorescent light. Among the most important advances were the
application of interference filters and the dichroic mirrors and their incorporation in a
versatile epi-illuminator (Ploem, 1967), the introduction of special high-NA objective
lenses, and the introduction of sensitive films and electronic imaging devices. Advances
continue today in all of these areas. Because of its great specificity and relative ease of
use, fluorescence microscopy is the most frequently employed mode of light micro-
scopy used in biomedical research today. 177