WOC 6e Guide to Microscopy

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Figure A-13 Fluorescence Microscopy. Cultured dog kidney
epithelial cells stained with the fluorescent stain phalloidin, which
binds to actin microfilaments.
fluorescence image of epithelial cells stained with a fluorescein-tagged
mushroom toxin, phalloidin, which binds specifically
to actin microfilaments. Another powerful fluorescence
technique utilizes the Green Fluorescent Protein (GFP), a naturally
fluorescent protein made by the jellyfish Aequoria victoria.
Using recombinant DNA techniques, scientists can fuse
DNA encoding GFP to a gene coding for a particular cellular
protein. The resulting recombinant DNA can then be introduced
into cells, where it is expressed to produce a fluorescent
version of the normal cellular protein. In many cases, the
fusion of GFP to the end of a protein does not interfere with its
function, allowing the use of fluorescence microscopy to view
the GFP-fusion protein as it functions in a living cell (Figure
A-14). Molecular biologists have produced mutated forms of
(a) 00:00 (b) 03:40 (c) 05:08
Figure A-14 Using Green Fluorescent Protein to Visualize Proteins. An image series of
a living, one-cell nematode worm embryo undergoing mitosis. The embryo is expressing
b-tubulin
that is tagged with the green fluorescent protein (GFP). Elapsed time
from the first frame is shown in minutes:seconds.
A-10 Appendix Principles and Techniques of Microscopy
GFP that absorb and emit light at a variety of wavelengths. In
addition, other naturally fluorescent proteins have been identified,
such as a red fluorescent protein from coral. These tools
have expanded the repertoire of fluorescent molecules at the
disposal of cell biologists.
In addition to detecting macromolecules such as proteins,
fluorescence microscopy can also be used to monitor
the subcellular distribution of various ions. To accomplish
this task, chemists have synthesized molecules whose fluorescent
properties are sensitive to the concentrations of ions
such as and Mg as well as to the electri-
2; Ca ,
2; , H ; , Na ; , Zn2; ,
cal potential across the plasma membrane. When these fluorescent
probes are injected into cells in a form that becomes
trapped in the cytosol or in a specific intracellular component,
they provide important information about the ionic
conditions inside the cell. For example, a fluorescent probe
called fura-2 is commonly used to track the Ca concentration
inside living cells, because fura-2 emits a yellow fluores-
2;
cence in the presence of low concentrations of and a
Ca 2;
green and then blue fluorescence in the presence of progressively
higher concentrations of this ion. Therefore, monitoring
the color of the fluorescence in living cells stained with
this probe allows scientists to observe changes in the intracellular
Ca concentration as they occur.
2;
CONFOCAL MICROSCOPY MINIMIZES BLURRING BY
EXCLUDING OUT-OF-FOCUS LIGHT FROM AN IMAGE
When intact cells are viewed, the resolution of fluorescence
microscopy is limited by the fact that although fluorescence
is emitted throughout the entire depth of the specimen, the
viewer can focus the objective lens on only a single plane at
any given time. As a result, light emitted from regions of the
specimen above and below the focal plane cause a blurring of
the image (Figure A-15a). To overcome this problem, cell
biologists often turn to the confocal microscope—a specialized
type of light microscope that employs a laser beam to
produce an image of a single plane of the specimen at a time
(Figure A-15b). This approach improves the resolution along
the optical axis of the microscope—that is, structures in the
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