Spectral unmixing procedures have been established to improve the image quality of multi-stained fluorescence samples. Based on the known spectral shape of different chromophores their relative contributions to the overall fluorescence emission can be derived. Thus a separation of sub-structures containing chromophores with overlapping emission spectra becomes possible if a multi-detector assembly is used. However, energy tansfer processes between emitting species can influence the results significantly. In this work we will present an approach which uses both time- and spectral information to discriminate dynamic processes from simple concentration effects in fluorescence imaging measurements. Our instrumentation covers a combination of a TCSPC based multi-wavelength detection technique together with a laser scanning microscope. The general solution obtained for two emitters exchanging energy by excited state transfer will be discussed.

© 2004 Optical Society of America

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