FCS cell surface measurements - Photophysical limitations and consequences on molecular ensembles with heterogenic mobilities
2005 (English)In: Cytometry Part A, ISSN 1552-4922, Vol. 68A, no 2, 101-112 p.Article in journal (Refereed) Published
Background: 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.
Place, publisher, year, edition, pages
2005. Vol. 68A, no 2, 101-112 p.
fluorescence correlation spectroscopy, photobleaching, diffusion, cell membrane, fluorescence correlation spectroscopy, living cells, correlation microscopy, 2-photon excitation, lateral diffusion, single molecules, c-peptide, membranes, dynamics, sensitivity
IdentifiersURN: urn:nbn:se:kth:diva-15191DOI: 10.1002/cyto.a.20193ISI: 000233456200005ScopusID: 2-s2.0-28244446665OAI: oai:DiVA.org:kth-15191DiVA: diva2:333232
QC 20100525 QC 201203012010-08-052010-08-052012-03-01Bibliographically approved