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  Fluorescence Correlation Microscopy of cells in the presence of autofluorescence

Brock, R., Hink, M. A., & Jovin, T. M. (1998). Fluorescence Correlation Microscopy of cells in the presence of autofluorescence. Biophysical Journal, 75, 2547-2557.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-FC6E-E Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EC02-F
Genre: Journal Article

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 Creators:
Brock, R.1, Author              
Hink, M. A., Author
Jovin, T. M.1, Author              
Affiliations:
1Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              

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Free keywords: Animal; Carbocyanines/me [Metabolism]; Dextrans/me [Metabolism]; Diffusion; Fluorescent Dyes/me [Metabolism]; Human; Lasers; Mice; Microinjections; *Microscopy, Fluorescence/mt [Methods]; Mitochondria/me [Metabolism]; Support, Non-U.S. Gov't; Time Factors; Tumor Cells, Cultured 3T3 Cells
 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.

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Language(s): eng - English
 Dates: 2005-07-081998
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: eDoc: 223392
Other: 621
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 75 Sequence Number: - Start / End Page: 2547 - 2557 Identifier: -