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Optogenetics measured by total internal reflection fluorescence fluctuations

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Optogenetics are recently developed tools that enables the use of light to activate or inhibit specific proteins and control intracellular processes. One key benefit of optogenetics is the capability to induce signalling perturbations that are localized in space and time, while classical genetic and pharmacologic approaches can only create permanent and global perturbations. Optogenetics is a particularly interesting tool for studying the spatio-temporal processes involved in cell signalling which governs cell behaviour during e.g. migration or division. Therefore, it is important to quantify the molecular activity following light irradiation and understand the physical processes that determines the spatial and temporal resolution of the induced effect.

In this project, the optogenetic system Cry2/CIBN will be studied in live cells. Upon blue light absorption, Cry2 associates with CIBN which is anchored at the cell membrane. We will focus on quantifying the recruitment of Cry2 molecules using image correlation spectroscopy (ICS), a family of methods based on the analysis of pixels’ intensity fluctuations in an image series. These fluctuations arise from changes in the occupation number of fluorophores in the focal volume. ICS can provide quantitative maps of protein density and diffusion constant of fluorescent molecules, by computing correlation functions in subregions of the image. In order to remove the fluorescence signal from out-of-focus planes in the cell, a total internal reflection (TIRF) configuration will be used : as the incident beam is translated to the edge of the objective pupil, the incident angle increases until the critical value that forbids propagation into the sample. Since the evanescent wave only extends a few hundreds of nanometers inside the sample, only fluorescent molecules attached to the membrane would be visible.
The proposed work would include first building the optical setup around an existing microscope. This setup will consist of 488 nm-and 561 nm-emitting lasers, a high NA objective and a fast camera. Then, the concentration, mobility and interactions of Cry2, CIBN and the photo-generated dimer will be measured as a function of irradiation intensity and duration. Based on these results, it would be possible to optimize the illumination conditions to improve the spatial resolution of light-induced effects.

Contact : Irène Wang – tél. 04 76 51 47 29 –
Antoine Delon – tél. 04 76 63 58 01–