Fluorescence correlation microscopy of cells in the presence of autofluorescence

被引:114
作者
Brock, R [1 ]
Hink, MA [1 ]
Jovin, TM [1 ]
机构
[1] Max Planck Inst Biophys Chem, Dept Biol Mol, D-37077 Gottingen, Germany
关键词
D O I
10.1016/S0006-3495(98)77699-4
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mel. 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) cm(2)/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.
引用
收藏
页码:2547 / 2557
页数:11
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