Optogenetics are recently developed tools, based on photosensitive proteins, 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.
Cry2/CIBN optigenetic system : the recruitment of Cry2 (labelled by the fluorophore mCherry) following optical stimulation is imaged in a confocal microscope.
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 (see figure). By fusing Cry2 with signalling proteins, it would be possible to activate specific cell signalling pathways. Here, we 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.
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