Kis-Petikova, K., Y. Chen, J. D. Müeller, and E. Gratton
Two-photon fluorescence correlation spectroscopy (FCS) has been proved as a powerful experimental technique for the study of fluctuations in the fluorescence signal of samples with low concentrations1. From the measured autocorrelation function, the diffusion coefficient, chemical reaction rates or number of labeled particles can be determined3.
In the scanning FCS (S-FCS) technique the exciting beam is scanned periodically across the sample. By scanning through independent subvolumes, sensitivity of the measurement can be improved significantly2,4. In addition, the autocorrelation function obtained from S-FCS in principle contains more information concerning the shape of diffusing molecules, when particles are of the size comparable to the laser beam size5 and scanning radius. Besides of time correlation, analyzed in FCS measurement, a spatial correlation at short distances can be analyzed. In the presented work, computer simulations of FCS and scanning FCS measurements of different size of particles were performed. Optimal scanning parameters, as diameter of the circular path, and angular frequency, were found to differentiate between particle shapes. Model systems were measured and analyzed, and advantages of scanning were demonstrated.
Supported by the NIH , PHS 5 P41 RR03155
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