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Principles and Technique of Fluorescence Microscopy


Interest in fluorescence microscopy has greatly increased in recent years. Technical considerations have to some extent prevented even wider application of the various fluorescence techniques now available for microscopical study of biological specimens. This paper outlines the basic requirements for optimal image quality, for the benefit of biologists and others who may not be conversant with the optical principles involved. The central problem of illumination is reviewed in some detail, and an assessment given of the two methods in current use, namely the bright-field and dark-field systems. Ratios of fluorescent to activating light received by the objective aperture, given by the two systems, have been compared, and measurements have been made of their relative light-concentrating power.

Available light sources and their suitability for the excitation of fluorescence are discussed, with the problems of selecting appropriate light filters for use with the alternative systems of illumination. It is concluded that the dark-field system has decided advantages in practice and in theory for the following reasons:

(1) The dark-field condenser serves as an efficient primary filter, contributing to a black background and hence good contrast.

(2) The equivalent focal length is less than that of the bright-field condenser and it concentrates energy in a smaller area; this compensates in part for the loss of energy inevitably caused by the central stop.

(3) It permits the use of wide-band primary filters of maximum transmission because contrast in the fluorescent image is affected only by a weak superimposed dark-field image produced in the object-plane by scattered residual activating light passed by the primary filter. With blue-light activation the visible dark-field image is effectively eliminated by means of a weak blue-absorbing secondary filter.

(4) The loss of contrast due to veiling glare is minimized.

A rational layout for fluorescence microscopy and methods for accurate alignment of the microscope in the vertical and horizontal positions are described. Factors influencing the choice of suitable objectives and eyepieces and some details of methods for mounting specimens are given.