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 – firstname.lastname@example.org
Antoine Delon – tél. 04 76 63 58 01– email@example.com