Single point FCS and number and brightness analysis on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)

Abstract

Fluorescence Correlation Spectroscopy is a technique developed in the early 1970s to measure chemical reaction rates, diffusion coefficient and photo physical processes. Commonly single point FCS data are obtained on sophisticated FCS instruments operating in the photon counting mode. Here we show that accurate FCS data can be obtained on a low cost commercial confocal laser scanning microscope without any modifications. Using the Nikon C1, we measured the diffusion coefficient and the concentration of Rhodamine B in solution for concentrations ranging from 5 nM to 280 nM. We also determined the diffusion coefficient of two different labeled lipid analogs (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and BODIPY TMR phosphatidylinositol (4,5) bisphosphate) incorporated into the membrane of giant unilamellar vesicles. The results obtained for these lipid analogs are in good agreementwith previously published data. Although single point FCS is possible with analog detectors the photon counting histogramanalysis is not. Number and brightness analysis is an alternative to PCH to determine the aggregation state of molecules. We show that the molecules incorporated into the GUV membrane are uniformly distributed into the membrane of the GUV. Finally, we highlight the fact that the actual proportion of labeled lipid analogs incorporated into the membrane of the giant unilamellar vesicle (formed by the electroformation method) is significantly different than the proportion of these lipids in the organic solvent stock solution.