FCS cell surface measurements--photophysical limitations and consequences on molecular ensembles with heterogenic mobilities.
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.


METHODS
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 photo-bleached. 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.
Widengren
Jerker
J
Department of Physics, Division of Experimental Biomolecular Physics, Royal Institute of Technology, Albanova University Center, 10691 Stockholm, Sweden. jerker@biomolphysics.kth.se
Thyberg
Per
P
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D004058:Diffusion; D004815:Epidermal Growth Factor / Q000032:analysis; D005456:Fluorescent Dyes; D008433:Mathematics; D038761:Photobleaching; D051381:Rats; D012235:Rhodamines / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D001749:Urinary Bladder Neoplasms / Q000737:chemistry / Q000473:pathology
D005456:Fluorescent Dyes; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate; D004815:Epidermal Growth Factor