ELIMINATION OF AUTOFLUORESCENCE IN IMMUNOFLUORESCENCE MICROSCOPY WITHDIGITAL IMAGE-PROCESSING

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