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Nanobiophotonics

 

 

Leader: Rodolphe Jafiol

 

Members :

Rodolphe Jaffiol, Cyrille Vézy, Jérôme Plain

 

Current studies:

Since 2005, we work on Fluorescence Correlation Spectroscopy (FCS) and its applications in biophysics, particularly membrane microfluidity in living cells.

In FCS, a sample of fluorophores located in the plasma membrane of living cells, is illuminated with a focused laser beam. In the "usual" approach of FCS, the fluctuations of the fluorescence signal are monitored while fluorophores are diffusing through the observation volume, which is restricted by a confocal detection (see setup below). Interestingly, significant amplitude fluctuations only arise from sufficiently low molecule concentrations, typically at the nanomolar concentration scale. Those required dilution conditions are a real advantage, especially for biological investigations, since they will create only slight perturbations in the sample, making FCS a quite non-invasive technique in regards to in vivo analysis.

The fluorescence signal F(t) is recorded using an avalanche photodiode (see schematic setup) and the fluorescence fluctuations are analyzed through the temporal autocorrelation function of F(t), namely the autocorrelation function denoted G(τ). The G(τ) decay time, namely the diffusion-time noted τd, corresponds to the average time required for the fluorescent molecules to cross the detection volume. For molecules diffusing within the plasma membrane, this diffusion-time is strongly influenced by the local organization and the heterogeneities of the membrane. Thus, reciprocally, FCS is well suited to retrieve micro-organization of cells membrane.

 

Tumoral cells could present a Multidrug Resistance (MDR) to chemotherapeutic treatments.  This drug resistance is mainly dependent on biomechanisms occurring at the plasma membrane level, and it would be associated to membrane-mediated mechanisms involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae), and proteins over expression. All these membrane mechanisms can be monitored by FCS through the lateral diffusion of a fluorescent probe. Consequently, FCS has been used to explore plasma membrane fluidity of multidrug-resistant cancer cells in comparison with the non-resistant ones (denoted sensitive cells), and also to characterize the influence of membrane agents present in extra-cellular medium (fluidity modulator and revertants of drug resistance).
For each type of cell-populations (i.e. resistant cells, sensitive cells,...) a statistical analysis of the diffusion-times recorded at the single cell level, have been performed. Two examples of diffusion-time distributions are presented below. Various different informations can be extracted from the diffusion-time distribution analysis. The average value gives the global fluidity of the plasma membrane. The standard deviation gives important informations about system heterogeneity. And slow diffusion-time measurements point out slow diffusion sites such as lipid microdomains. Such method, when conducted on a large number of living cells, can enable us to get a detailed overview of the plasma membrane microviscosity, and consequently micro-organization.

 

Collaborations:

J-M. Millot et P. Jeannesson, Unité MEDyC (Matrice Extracellulaire Dynamique Cellulaire), UMR CNRS 6237, Faculté de Pharmacie, Reims, France.

 

Recent related publications:

[1] Surface modified single molecules free-diffusion evidenced by fluorescence correlation spectroscopy, C. Boutin, R. Jaffiol, J. Plain, P. Royer, Journal of Fluorescence (2008) in press (on line).

[2] Effect of different agents onto multidrug resistant tumor cells revealed by fluorescence correlation spectroscopy, C. Boutin, Y. Roche, C. Millot, R. Deturche, J. Plain, P. Jeannesson, M. Manfait, P. Royer, J-M. Millot, R. Jaffiol, Spectroscopy (2008) in press.