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 conduct 3 main activities :
We are developing cryogenic temperature PALM microscopy (cryo-PALM), which is one of the major current advancements in the field, particularly for applications in integrated structural biology based on correlative microscopy (cryo-CLEM). For this project, we benefit from a cryo-microscope developed by Jörg Enderlein’s German team (University of Göttingen). Our goal is to study the photocommutation properties of PTFPs at low temperatures, to engineer fluorescent proteins better suited for cryo-PALM, or to improve laser illumination protocols during cryo-PALM data collection. For example, we recently discovered a new photocommutation mechanism occurring at cryogenic temperatures in rsEGFP2 and showed that illumination at 355 nm increased labeling efficiency.
We study how the photophysics of photoconvertible fluorescent proteins, such as mEos variants, affect qPALM and sptPALM techniques. For example, we found a method to increase the length of traces in sptPALM with mEos variants by adding low illumination at 488 nm during data collection. We also recently showed that, due to a non-linear photobleaching mechanism, using high intensities of 405 nm light can be very detrimental to labeling efficiency in qPALM. Finally, our work in collaboration with the NMR group at IBS revealed the existence of multiple conformational states in mEos proteins like mEos4b, both in their green and red states, with consequences for photoconversion and blinking mechanisms in PALM experiments.
We are developing a single-molecule imaging simulator called SMIS, which, unlike all other simulators available to date, integrates an advanced description of the spectral and photophysical properties of fluorophores. SMIS enables complex simulations that can predict, for example, the effects of various illumination conditions in a PALM experiment or the subtle effects of crosstalk in multicolor SMLM experiments.