Castellini, Horacio(1); Dumas Dominique(2); Riquelme, Bibiana(3)
- Dpto Física, Fac Cs Exactas Ingeniería y Agrimensura, UNR.
- Lab Ing Celular y Tisular, UHP, VandoeuvrelèsNancy, France.
- Física, FCByF, UNR. Bio-Optica., IFIR (CONICET-UNR).
The molecular diffusion in biological membranes is a determining factor in cellular function. Fluorescence Correlation Spectroscopy (FCS) technique has become the most important technique in microscopy field because his versatility to analyze the molecular dynamics in both biological and artificial membranes. It is based on the quantification of fluorescence intensity fluctuations emitted by a few small numbers of particles in a small volume (1 fL) at nanoseconds time scales. This signal is processing with the autocorrelation function and analytical expressions are fitting and it is allow interest kinetic parameters obtained, such as time and the diffusion coefficient. In this study we have developed a genetic algorithm in PDL (Perl Data Language) that allows fitting relevant parameters in cell biology using the FCS signal. The calibration and technique tuning conducted experiments in FCS with Rhodamine B dilute solutions (3 nM to 900 nM) in ultrapure water using two different lighting conditions at 543nm wavelength determined by the HeNe laser power (12% and 51%). We determined the effective volumes size (Veff 12% = (0.490.01) fL and Veff 51% = (0.620.01) fl) and the diffusion coefficient for the Rhodamina B in aqueous solution, whose value is keeping with the literature (3 x 106cm2/s). One can conclude that the accuracy on certain parameters is satisfactory, being an advantage for studies in the FCS can to have a program developed in open source freely accessible to the scientific community.