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Elimination of autofluorescence in immunofluorescence microscopy with digital image processing

CH Van de Lest, EM Versteeg, JH Veerkamp and TH Van Kuppevelt

Department of Biochemistry, University of Nijmegen, The Netherlands.

Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluorescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a self-developed computer program, results in an autofluorescence-free image. The procedure is demonstrated for elimination of elastin-derived autofluorescence in human lung alveoli and for elimination of lipofuscin-derived autofluorescence in human heart muscle. The autofluorescence signal is positively correlated with tissue section thickness (r = 0.93; p < 0.0001), and can be used to correct the specific fluorescence signals for section thickness.

Volume 43, Issue 7, pp. 727-730, 07/01/1995
Copyright © 1995 by The Histochemical Society


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