FLUORESCENCE CORRELATION MICROSCOPY OF CELLS IN THE PRESENCE OF AUTOFLUORESCENCE

Fluorescence correlation microscopy (FCM), the combination of fluoresc ence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mel. Biol. 44:847-856), has been implemented for me asuring molecular diffusion and association in living cells with expli cit consideration of autocorrelations arising from autofluorescence. A utofluorescence excited at 532 nm colocalizes with mitochondria, has f lavin-like spectral characteristics, exhibits relaxation times charact eristic for the diffusion of high-molecular-weight proteins, and depen ds on the incubation conditions of the cells. These time- and location -dependent properties preclude the assignment of universal background parameters. The lower limit for detection of microinjected dextran mol ecules labeled with the carboxymethylindocyanine dye Cy3 was a few tho usand molecules per cell, and the diffusion constant of 1.7 x 10(-7) c m(2)/s agreed well with values measured with other methods. Based on t he fluorescence signal per molecule (fpm) and the molecule number deri ved from autocorrelation analysis, a new method is devised to define i ntracellular association states. We conclude that FCM is a powerful, n oninvasive method for probing molecular interactions in femtoliter vol ume elements within defined subcellular locations in living cells.