Fluorescence Microscopy

 Fluorescence microscopy uses the contrast created by fluorescent molecules, fluorophores, that emit light of a specific wavelength when excited by incident light of a different (shorter) wavelength. By using filters, one can select an appropriate wavelength for excitation of the fluorophore and a second filter can eliminate light of wavelengths other than that emitted by the fluorophore. The second filter is very important as it removes the much more intense excitation light, so that the emitted signal is detected in the absence of the excitation light and other stray light, giving fluorescence microscopy an excellent signal-to-noise ratio. Fluorescence microscopy universally uses epi-illumination in which light from above is reflected off the surface of the specimen by light emerging from the objective lens. Epi-illumination has the advantages that (1) unabsorbed light is directed away from the observer and (2) the alignment is simplified since the objective lens acts as its own condenser. It is important to understand the capabilities and limitations of the fluorophores. Because a specimen can be stained with a finite quantity of fluorescent dye, which decays or photobleaches when illuminated, the microscope must be configured to efficiently excite and detect the limited number of emitted photons before complete bleaching has occurred. Although a large number of fluorescent dyes have been discovered, relatively few are suitable for fluorescence microscopy (1).

Immunofluorescence light microscopy is the most common technique of fluorescence microscopy and involves the use of antibodies labeled with fluorophores to visualize distributions of proteins and nucleic acids within a specimen. This technique can be performed by either direct or indirect antibody labeling. Direct immunofluorescence requires the conjugation of a primary antibody with a fluorophore, commonly rhodamine and fluorescein. Indirect immunofluorescence, on the other hand, uses a fluorophore conjugated secondary antibody which was raised against the immunoglobulin type of the primary antibody. For example, if the primary antibody is raised in a rabbit, the secondary antibody might be a goat anti-rabbit antibody. Indirect immunofluorescence is the more common of the two techniques because making a fluorophore conjugate of each primary antibody is time-consuming and can decrease the affinity or specificity for the antigen. Furthermore, the indirect technique may be more sensitive because more than one molecule of the secondary antibody may bind to a molecule of the primary antibody (2), creating an amplification of the signal. Indirect immunofluorescence has motivated the development of a large number of commercially available secondary antibodies conjugated to various fluorophores, so in order to do immunofluorescence microscopy, one need only raise and characterize the primary antibody and then purchase the secondary antibody. A key consideration in immunofluorescence microscopy is confirmation of the specificity of both fluorophore and antibodies. In order for immunofluorescence results to be validly interpreted, one must demonstrate that (i) the secondary antibody recognizes only the primary antibody, and (ii) the primary antibody recognizes only the antigen. This means that the secondary antibodies must not cross-react with intrinsic proteins and do not recognize each other. It is also necessary to establish whether autofluorescence from compounds intrinsic to the tissue can mimic the appearance of fluorophores. This test is easily done by examining an unstained specimen using the same excitation and emission filters intended for use with the fluorophore.

 Fluorescence microscopy can also be used to monitor intracellular compartments through the use of fluorescent indicator dyes, which report on their local environments. Some of the fluorescent dyes useful for microscopy are membrane potential indicators such as JC-1 (3). Using this probe, it was shown that heterogeneity in mitochondrial membrane potential exists between different mitochondria in the same cell (4). Other fluorescent dyes are ion indicators, which can report the pH or concentrations of calcium, chloride, magnesium, potassium, and sodium ions either qualitatively or quantitatively (5).

With the appropriate choice of fluorescent labels and proper equipment, one can do multilabeling [sometimes called multicolor (6)] to localize two or more molecular species using fluorophores with different excitation and/or emission wavelengths. By using multicolor immunofluorescence microscopy, the neurotransmitters, serotonin and substance P, were observed in separate populations of spinal cord fibers (6) and the motor neurons and the substance P fibers, which wrap around them, could be separately visualized. Another example is the use of DiOC6 to label mitochondria and ethidium bromide to label mitochondrial DNA in Euglena gracilis cells (7).

References

1. R. Y. Tsien and A. Waggoner (1990) In Handbook of Biological Confocal Microscopy, 2nd ed.)J. Pawley, ed.), Plenum Press, New York, pp. 169–178. 

2. L. A. Sternberger (1986) Immunocytochemistry, 3rd ed., Wiley, New York. 

3. M. Reers, T. W. Smith, and L. B. Chen (1991) Biochem. 30, 4480–4486. 

4. S. T. Smiley et al. (1991) Proc. Natl. Acad. Sci. 88, 3671–3675. 

5. B. Herman and J. J. Lemasters (1993) Optical Microscopy, Academic Press, San Diego. 

6. T. C. Brelje, M. W. Wessendorf, and R. L. Sorenson (1993) "Multicolor laser scanning confocal immunofluorescence microscopy: Practical application and limitations", In Methods in Cell Biology, Vol. 38: Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.), Academic Press, San Diego, pp. 98–182. 

7. D. L. Spector, R. D. Goldman, and L. A. Leihwand (1998) Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, New York.