Fluorescence correlation microscopy of cells in the presence of autofluorescence.
Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mol. Biol. 44:847-856), has been implemented for measuring molecular diffusion and association in living cells with explicit consideration of autocorrelations arising from autofluorescence. Autofluorescence excited at 532 nm colocalizes with mitochondria, has flavin-like spectral characteristics, exhibits relaxation times characteristic for the diffusion of high-molecular-weight proteins, and depends 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 molecules labeled with the carboxymethylindocyanine dye Cy3 was a few thousand molecules per cell, and the diffusion constant of 1.7 x 10(-7) cm2/s agreed well with values measured with other methods. Based on the fluorescence signal per molecule (fpm) and the molecule number derived from autocorrelation analysis, a new method is devised to define intracellular association states. We conclude that FCM is a powerful, noninvasive method for probing molecular interactions in femtoliter volume elements within defined subcellular locations in living cells.
Brock
R
R
Department of Molecular Biology, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
Hink
M A
MA
Jovin
T M
TM
D016475:3T3 Cells; D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D004058:Diffusion; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007834:Lasers; D051379:Mice; D008845:Microinjections; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D003911:Dextrans; D005456:Fluorescent Dyes; C089187:cyanine dye 3