JCJC - Jeunes chercheuses & jeunes chercheurs

Nouvelles méthodes d'analyse locale par FCS : dynamique membranaire de cellules tumorales – LocalFCS

Submission summary

1) Scientific background and objectives: In the early 70's, American researchers have offered to the fluorescence microscopy community a new spectroscopy technique based on a statistical analysis of the emission of fluorophores in solution: the Fluorescence Correlation Spectroscopy (FCS). The observed fluorescence fluctuations are related to the mean number and the diffusion velocity of the molecules present in the probing volume. The biophysical applications of FCS are numerous. One can for example study the mobility, the molecular interactions or also monitor conformational changes. However, until now the experimental conditions required for the FCS to be set up are very restrictive, limiting its use to ultra pure biomimetic systems and excluding de facto any kind of in vivo studies. In our project, we offer to develop an optical technique enabling us to overcome those limitations and thus allowing us to subsequently investigate real systems. Through a tight collaboration with a biophysicist group, the young "Nanobiophotonic" team of the LNIO will devote its experimental progress to the study of membrane dynamic of tumor cells. For this purpose, we will analyze the membrane diffusion of living cells by combining FCS with an original microscopy technique, dissociating our approach from those currently offered in the FCS community. This latter technique aims to significantly decrease the probing volume, via an indirect and highly localized excitation involving energy transfer, and efficiently setting us free from the strong heterogeneities of the plasma membrane. This original FCS implementation will enable us to prospect a large number of biological mechanisms leading to, and thus characterized, by modifications of the cohesion and the structure of the cellular membrane. 2) Project description and methods: This research project sits on a tight collaboration between the physicist bearer team, settled at the UTT, and a biophysicist one in the URCA. This project takes its roots in a significant manner at the interface between biology and instrumental physics, reflecting the interdisciplinary will to proceed through knowledge at ever smaller scales. At first the experimental development we propose consists in the combination of FCS with near-field optical microscopy. This latter allows to visualize spatially confined optical signals thanks to a nanometric size probe. The goal pursued in this project is to develop a nano-source of light allowing to perform FCS in a "nanometric" volume (typically a few attoliter, or below). The experimental improvements we propose to apply to the FCS setup have never been considered so far: we propose to perform the excitation of the dye molecule not directly by the laser source, but through a non-radiative transfer process, known under its acronym: FRET (Förster Resonance Energy Transfer). In regards to the nanometric range of such transfer process, this kind of FRET-illumination will provide a chemical selectivity together with an increased spatial resolution, thus allowing us to analyze finely the membrane dynamic. We will then use the original capacities of our improved setup to study phenomena in samples of biological interest. In the cancer research field, drug resistance to chemotherapy is mainly related to plasma membrane biochemical processes. The drug interactions with the membrane modulate the chemoresistance through membrane-mediated mechanisms involving fluidity, permeability, presence of microdomains (rafts, caveolae) and variation of membrane proteins expression. In this project, we propose to analyze, thanks to our optimized microscope, and at the single cell level, some of these membrane mechanisms. This should allow us to get a new insight on some of the fundamental mechanisms of drug resistances related to plasma membrane processes. 3) Main expected results: The main results to be sought after in this project are the following one: -The demonstration of a strong reduction of the optical probing volume thanks to the FRET nano-illumination source. -At the single cell level, the deciphering of the membrane fingerprints influencing drugs resistance via a better knowledge of the plasma membrane organization. In regards to the forthcoming innovative outcomes to be expected at long or middle term, as well as the already significant contribution to be reached through the intermediate stages, we expect to quickly spread our results in scientific journals and conferences of the different communities, covering thus many different fields, from high resolution optical microscopy to cell biology, via surfaces and interfaces physical-chemistry, fluorescence spectroscopy...

Project coordination

Rodolphe JAFFIOL (Université)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Help of the ANR 151,000 euros
Beginning and duration of the scientific project: - 36 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter