Cécile Fradin, Asmahan Abu-Arish, David Zbaida,. Rony Granek and Michael Elbaum. Department of Materials & Interfaces, Weizmann Institute of Science, ...
Study of the elastic properties of a membrane using fluorescence correlation spectroscopy Cécile Fradin, Asmahan Abu-Arish, David Zbaida, Rony Granek and Michael Elbaum Department of Materials & Interfaces, Weizmann Institute of Science, Rehovot, Israel Fluorescence correlation spectroscopy (FCS) is a method which allows to study the dynamics of phenomenon causing fluctuations in the amount of fluorescence collected from a confocal volume by computing the autocorrelation function of this signal [1,2]. Because it is sensitive to single molecules, has very good spatial and temporal resolutions (0.5 × 0.5 × 3 µm and 12 ns respectively), and is non invasive and non destructive, there is currently a growing interest in applying FCS to the in vivo study of biological systems [3-5]. Nevertheless, the problem of the crowding of the cell environment, which causes the appearence of poorly understood long-time correlations in the autocorrelation function, makes it difficult to interpret the results obtained [5]. In order to clarify the influence of the presence of the different cellular membranes, we studied, both theoretically and experimentally, the autocorrelation function obtained in the presence of a single vertical membrane. In the case of a single fluorophore freely diffusing in solution, the autocorrelation function simply exhibits a characteristic decay time corresponding to the residence time of the particles in the confocal volume, and inversely proportionnal to the diffusion coefficient D of the particles. We showed that if a membrane is placed close to the confocal volume, the autocorrelation function will be affected in two ways. Firstly, since the diffusion of the particles is modified by the membrane, the characteristic decay time of the diffusion term changes, leading to erroneous estimations of D if the presence of the membrane is not acknowledged. Secondly, if the membrane is soft, its thermal undulations will lead to fluctuations in the effective detection volume, and hence one needs to add a new term in the autocorrelation functions, with an amplitude inversely proportionnal to the bending rigidity K of the membrane.
Figure 1: Autocorrelation functions measured in presence of a lipid bilayer membrane, far from the membrane (black dots), and 100 nm (empty circles), 65 nm (grey triangles), and 20 nm (empty squares) away from the membrane. The continuous lines are fit of the diffusion term by the usual function calculated for free diffusion.
To check these predictions, we did FCS measurements close to the lipid bilayer of a DOPC vesicle, after adding 10 nM of fluorescent strepavidin outside the vesicle. Fig. 1 shows a few different autocorrelation functions obtained, for different distance of the detection volume to the wall. Far away from the membrane, the autocorrelation function can be accounted for by simple 3D diffusion of the streptavidin molecules. Close to the membrane, one can observe the modification in shape of the diffusion term, and the contribution of the thermal fluctuations. The measured amplitudes of the diffusion term and of the membrane undulation term (Fig. 2a and 2b) are in very good agreement with our calculations, and allow to estimate the bending rigidity of the membrane to about 400 kT.
Figure 2: a-Measured amplitude of the diffusion term (dots). The best fit using our theory (continuous line) gives a signal over noise ratio of about 5. b-Measured amplitude of the membrane fluctuations term (dots). The best fit using our theory gives a bending rigidity for the membrane K=470 kT. Both curves are given in fuction of the normalised intensity (equal to 0 inside the vesicle and far away from the membrane, and equal to 1 outside the vesicle and far away from the membrane). Our results suggest that some of the long-time correlations observed in the autocorrelation functions measured by FCS in cells might be due to the presence of cellular membranes close to the detection volume. This would lead to erroneous values of the the measured diffusion coefficients of fluorescent particles. On the other hand, we found that FCS allows to observe the thermal fluctuations of membrane, which might provide a new method to measure bending rigidities in vivo, and with a very good spatial resolution.
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5. M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy”, J. Mol. Biol. 298:677-689, 2000.
