Certain dyes, called fluors or fluorochromes, can be raised to a higher energy level after absorbing ultraviolet (excitation) light. When the dye molecules return to their normal, lower energy state, they release excess energy in the form of visible (fluorescent) light. This process is called fluorescence, and microscopic methods have been developed to exploit the enhanced contrast and detection that this phenomenon provides.
Figure 6-11 depicts diagrammatically the principle of fluorescent microscopy in which the excitation light is emitted from above (epifluorescence). An excitation filter passes light of the desired wavelength to excite the fluorochrome that has been used to stain the specimen.
A barrier filter in the objective lens prevents the excitation wavelengths from damaging the eyes of the observer. When observed through the ocular lens, fluorescing objects appear brightly lit against a dark background.
The color of the fluorescent light depends on the dye and light filters used. For example, use of the fluorescent dyes acridine orange, auramine, and fluorescein isothiocyanate (FTTC) requires blue excitation light, exciter filters that select for light in the 450- to 490-X wavelength range, and a barrier filter for 515 X. Calcofluor white, on the other hand, requires violet excitation light, an exciter filter that selects for light in the 355- to 425-X wavelength range, and a barrier filter for 460 X. Which dye is used often depends on which organism is being sought and the fluorescent method used. The intensity of the contrast obtained
with fluorescent microscopy is an advantage it has over the use of chromogenic dyes (e.g., crystal violet and safranin of the Gram stain) and light microscopy.
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