Accueil > Research > Research groups > Integrated Imaging of Stress Response Group (Dominique Bourgeois) > Pixel
Pixel PresentationResponsable : Dominique Bourgeois
Presentation
Pixel is a multidisciplinary team that works at the frontier between structural and cell biology. The team was created in 2010, motivated by the evolution of modern structural biology towards integration into the cellular context. The Pixel team has a strong methodological focus based on two main objectives :
One of our central themes concerns photophysical studies of phototransformable fluorescent proteins (PTFPs). These genetically encoded fluorescence markers are fascinating because their fluorescence state can be tuned by illumination with proper laser light. For example, the emission color of some PTFPs can be changed from green to red upon illumination with violet light (so-called photoconvertible FPs, PCFPs). Other PTFPs can be reversibly switched from on states to off states by alternating cyan and violet light (so-called reversibly photoswitchable FPs, RSFPs). PTFPs are fundamental players in super-resolution microscopy as well as other advanced fluorescence methods such as pulse-chase imaging, modulated-illumination imaging, photochromic Förster Resonance Energy Transfer (FRET) or biological data storage. Phototransformation properties like photoactivation, photoconversion and photoswitching need to be understood and optimized for each application. In addition, photoblinking (the stochastic and transient loss of fluorescence) and photobleaching (the irreversible loss of fluorescence) are crucial photophysical properties that apply to all fluorescent proteins and that we investigate in great details, although they still hide a lot of mysteries. In the last years, we have largely focused our attention on photoblinking and photobleaching in popular PCFPs such as mEos4b. These photophysical processes seem to exhibit a never ending complexity, but we discover more and more about them. For example, in collaboration with the team of P. Dedecker (KUL, Belgium) we recently deciphered a major mechanism of blinking in red mEos4b, typically causing serious trouble in the quality and interpretation of quantitative PALM (qPALM) or sptPALM data, and showed that this mechanism very closely relates to the reversible photoswitching mechanisms at play in RSFPs. This allowed us to propose a trick to reduce blinking in sptPALM, so as to enable the reconstruction of significantly longer tracks. We also turned our attention to green state photophysics in PCFPs. Indeed, although PCFPs are only detected once photoconverted to the red state, what happens before photoconversion considerably affects their performance as markers for SMLM. Recently, in collaboration with the teams of J.B Sibarita and M. Sainlos (IINS, Bordeaux), we started a project aiming at further improving the photostability of PCFPs, by combining structural studies with high-content-screening single-molecule imaging approaches to achieve efficient semi-rational engineering. In parallel with these photophysical studies, 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. Recently, in collaboration with the german team of Jörg Enderlein (Göttingen University), we have started the development of PALM microscopy at cryogenic temperatures (cryo-PALM) which constitutes one of the major developments 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. CollaborationsExternal collaborations :
Internal Collaborations (Super-resolution microscopy : biological projects) :
Collaborations internes (Fluorescent proteins photophysics) :
StaffPermanent Staff :
Non-Permanent Staff :
PhD Students :
former Postdocs :
Former PhD Students :
Research topics
Key wordsFluorescent proteins ; super resolution fluorescence microscopy ; TIRF microscopy, single molecule imaging ; photoactivation ; blinking, photobleaching microspectrophotometry ; kinetic crystallography ; structural dynamics ; photophysics ; Protein dynamics and mechanisms ; Macromolecular nanomachines ; Methods developments ; Protein design and engineering. Specialized techniques
Highlights
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