In:
Cytometry Part A, Wiley, Vol. 68A, No. 2 ( 2005-12), p. 101-112
Abstract:
Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked. Method Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions. Results Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photobleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored. Conclusions Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding. © 2005 Wiley‐Liss, Inc.
Type of Medium:
Online Resource
ISSN:
1552-4922
,
1552-4930
DOI:
10.1002/cyto.a.v68a:2
DOI:
10.1002/cyto.a.20193
Language:
English
Publisher:
Wiley
Publication Date:
2005
detail.hit.zdb_id:
2180639-1
SSG:
12
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