Analytical Cytology: Methods for Studying Cellular Form and Function

By Robert C. Mellors | Go to book overview

CHAPTER 5 Fluorescence Microscopy

OSCAR W. RICHARDS, Ph.D. American Optical Company, Research Center, Southbridge, Mass.


Introduction

Fluorescent materials, when activated by the absorption of radiant energy, emit radiation at another (usually longer) wavelength on return to their previous energy state. Either invisible ultraviolet or visible blue-violet radiation is generally used in fluorescence microscopy to energize the specimen, which then becomes self-luminous, usually in color. Some materials have sufficient intrinsic or autofluorescence for direct visual examination; others can be made to fluoresce by impregnating them with fluorescent chemicals called fluorochromes or fluors.

Fluorescence microscopy evolved from Köhler's pioneer work with the ultraviolet microscope (96). Summaries of the method have been written by Lehman (99), Radley and Grant (149), Dhéré (38), Haitinger (70), Barnard and Welch (6, 7), Loos (113), Ellinger (44), Heyroth (79), Metcalf and Patton (127), Hamley and Sheard (71), Strugger (186), Braütigam and Grabner (18), Zeiss (202), and Richards (152). The 1939 Discussion of the Faraday Society, the work of Leverenz (107), and the monograph of Pringsheim (148) deal with the physical and theoretical aspects of fluorescence.


Equipment

MICROSCOPE

Optical glass transmits the long wavelength ultraviolet light that excites the fluorescence of most biological specimens. Therefore, the customary light microscope can be used for fluorescence microscopy. In some special problems, such as the study of mineral fluorescence which is activated by the shorter wavelength ultraviolet, quartz optics are required for the lens components of the illuminator and the microscope condenser. Since silver is not a good reflecting material for ultraviolet, Fig. 5·1, the substage mirror of the microscope is replaced by an aluminized, front-surface mirror. Slip-on mounts for these mirrors are available. Rhodium may be useful as a reflecting material (25).

-5/1-

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