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Biophysical Journal 84:2005-2020 (2003)
© 2003 The Biophysical Society

Fluorescence Correlation Spectroscopy Close to a Fluctuating Membrane

Cécile Fradin*,{dagger}, Asmahan Abu-Arish*, Rony Granek*,{ddagger} and Michael Elbaum*

* Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel; {dagger} Department of Physics and Astronomy and Department of Biochemistry, McMaster University, Hamilton, Ontario L8S 4M1, Canada; and {ddagger} Department of Biotechnology Engineering and the Institute for Applied Biosciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

Correspondence: Address reprint requests to Cécile Fradin, Dept. of Physics and Astronomy, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4M1, Canada. Tel.: 1-905-525-9140; Fax: 1-901-546-1252; E-mail: fradin{at}physics.mcmaster.ca.

Compartmentalization of the cytoplasm by membranes should have a strong influence on the diffusion of macromolecules inside a cell, and we have studied how this could be reflected in fluorescence correlation spectroscopy (FCS) experiments. We derived the autocorrelation function measured by FCS for fluorescent particles diffusing close to a soft membrane, and show it to be the sum of two contributions: short timescale correlations come from the diffusion of the particles (differing from free diffusion because of the presence of an obstacle), whereas long timescale correlations arise from fluctuations of the membrane itself (which create intensity fluctuations by modulating the number of detected particles). In the case of thermal fluctuations this second type of correlation depends on the elasticity of the membrane. To illustrate this calculation, we report the results of FCS experiments carried out close to a vesicle membrane. The measured autocorrelation functions display very distinctly the two expected contributions, and allow both to recover the diffusion coefficient of the fluorophore and to characterize the membrane fluctuations in term of a bending rigidity. Our results show that FCS measurements inside cells can lead to erroneous values of the diffusion coefficient if the influence of membranes is not recognized.




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