Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield



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Detecting Fluorochrome-Labeled Reagents

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The low levels of fluorescence produced in cell-staining experiments require a microscope equipped for epifluorescence, in which the exciting radiation is transmitted through the objective lens onto the surface of the specimen. Absorbing radiation of the appropriate wavelength causes the electrons of the fluorochrome to be raised to a higher energy level. As these electrons return to their ground state, light of a characteristic wavelength is emitted. This emitted light forms the fluorescent image seen in the microscope. Individual fluorochromes have discrete and characteristic excitation and emission spectra. Filters are used to ensure that the specimen is irradiated only with light at the correct wavelength for excitation. By placing a second set of filters in the viewing light path that only transmit light of the wavelength emitted by the fluorochrome, images are formed only by the emitted light. This produces a black background and a high-resolution image.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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