title: Fluorescence correlation microscopy of cells in the presence of autofluorescence.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Brock
authors.forename: M A
authors.initials: MA
authors.lastname: Hink
authors.forename: T M
authors.initials: TM
authors.lastname: Jovin
mesh_terms: 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
chemical_list: D002232:Carbocyanines; D003911:Dextrans; D005456:Fluorescent Dyes; C089187:cyanine dye 3