Developpement of qPALM and sptPALM microscopies

We develop PALM super-resolution microscopy, a single-molecule-based localization method now used worldwide to overcome the diffraction limit. PALM and its derivatives qPALM and sptPALM are almost entirely based on the proper manipulation of PTFP’s photophysics, which brings coherence to our activities.

Our PALM microscope is used to study PTFPs in cellulo (or in vitro) at the single molecule level. We perform 3 main activities:

 In collaboration with the german team of Jörg Enderlein (Göttingen University), we develop PALM microscopy at cryogenic temperatures (cryo-PALM) which constitutes one of the major evolution in the field in the years to come, especially for applications in integrated structural biology based on correlative microscopy (cryo-CLEM). Our role in this project is to investigate PTFP’s photoswitching capabilities at low temperature. We recently discovered a new photoswitching mechanism occurring at cryogenic temperature in rsEGFP2.

 We investigate how the photophysics of photoconvertible fluorescent proteins such as mEos variants affect qPALM and sptPALM quantitatively. For example, we found a way to increase track length in sptPALM using mEos variants by adding gentle 488 nm light during data collection. We showed recently that, due to a nonlinear photobleaching mechanism, using high intensities of 405 nm light can be very deleterious to the achieved effective labeling efficiency in qPALM.

 We develop a single molecule imaging simulator called SMIS which, contrary to all available simulators to date, incorporates an advanced description of fluorophore spectral and photophysical properties. SMIS allows performing complex simulations able to predict for example the effects of various illumination conditions in a PALM experiment, or subtle crosstalks effects in multicolor SMLM experiments.