Cell membrane investigations

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FCS investigations of cell membranes

Principal investigator : Patrick Ferrand, Jerome Wenger

Principle and Applications

Fluorescence Correlation Spectroscopy uses a confocal microscopy set-up to collect the light from luminescent molecules moving across an observation volume which can be as small as 0.2 fl (1fl=1µm3). The fluctuating number of fluorophores in this observation volume gives rise to intensity fluctuations which are detected by avalanche photodiodes. The fluorescence intensity is analyzed with a digital correlator which builds the temporal autocorrelation function (ACF). The ACF gives informations on diffusion, molecular concentration and associations. [1]

It has been demonstrated in our team that even sub-diffraction structures can be quantified using a multiscale analysis approach [2,3]. In order to overcome the inherent drawbacks of local measurements (bleaching due to the continuous exposure to the excitation, absence of control of drift, etc.), many variants have been proposed that use a scanned observation volume. Beyond the spatial information, they usually provide also a better flexibility in terms of timescale, a lower bleaching, and a better statistics. [4]

New instrumental developments

We are currently developing a fluorescence multipurpose system made by the
combination of two fully independent custom confocal laser scanning microscopes (CLSM) systems. Thus, two excitation spots can be generated independently within the three dimensions of the sample, each one being associated to a detection
channel. The system was designed in order to provide the highest versatility for a multimodal analysis. It can operate either two simultaneous independent single spot measurements (including CLSM imaging, FCS, Photon Counting Histogram, scanning FCS, etc.) or one real-time cross-analysis between two distant locations. [5] More recently, polarisation control and analysis was implemented, in order to monitor rotational diffusion of single molecules.

List of avalaible FCS systems

Three FCS systems have been developped by the MOSAIC team :

  • one single spot FCS system, optimized for FCS diffusion laws measurements, moved to the lab of our bioliogy partners (CIML), and routinely used there,
  • one single spot FCS system, optimized for nanoapertures measurements,
  • one dual spot FCS system, optimized for dual spot measurements [5], as well as fast confical imaging (see Figure)
DualFCS setup - Photo Patrick Ferrand

People involved

  • Patrick Ferrand
  • Alla Kress
  • Jerome Wenger


This project was funded by Agence Nationale de la Recherche under contract ANR-05-BLAN-0337-02 and Région Provence Alpes Côte d’Azur

Additionnal Ressources

Wikipedia article on FCS

What does FCS have to do with football/soccer ?


[1] For a review, see P. Schwille, Cell Biochem. Biophys., 34, 383, 2001.

[2] P. F. Lenne, L. Wawrezinieck, F. Conchonaud, O. Wurtz, A. Boned, X.-J. Guo,
H. Rigneault, H.-T. He, and D. Marguet, EMBO J., 25, 3245, 2006. - PDF

[3] L. Wawrezinieck, H. Rigneault, D. Marguet D., P.-F. Lenne P.-F., Biophys. J. 89, 4029, 2005. - PDF

[4] For a review, see D. L. Kolin, P. W. Wiseman, Cell Biochem. Biophys., 49,
141, 2007.

[5] P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, D. Marguet, "A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell", Review of Scientific Instruments 80, 083702 (2009) - PDF.