Blanc SVSE 5 - Sciences de la vie, de la santé et des écosystèmes : Physique, chimie du vivant et innovations biotechnologiques

Tunable Time-Resoved Stimulated Emission Depletion Microscopy for dynamic imaging of interactions at the subcellular scale. – STED-FLIM

STED microscopy

The goal of the project is to develop a microscope to break the diffraction limit and reveal Alzeiner disease mechanisms

Super-resolution microscopy for neurobiology

L’objectif est de développer un système de microscopie STED à super-résolution accordable en longueur d’onde afin de pourvoir exciter une large gamme de fluorophores d’intérêt biologique, voire plusieurs fluorophores simultanément. En outre, la mesure de la fluorescence pourra être effectuée de manière résolue en temps afin d’accéder aux durées de vie de fluorescence des fluorophores. En complément des informations de localisation à l’échelle nanométrique, une analyse dynamique de processus moléculaires et métaboliques au sein des cellules sera ainsi possible, ainsi qu’une colocalisation de deux protéines. Ceci permettra ainsi par exemple des études à l’échelle de la synapse jusqu’à présent inaccessibles. Ce système d’imagerie ouvrira ainsi de nouvelles perspectives dans l’étude de nombreux processus biologiques, notamment en neurobiologie (maladie d’Alzheimer), où les domaines d’interactions mis en jeu ne peuvent être spécifiquement étudiés aujourd’hui à cause d’une résolution spatiale insuffisante. Implanté sur la plate-forme d’imagerie du Centre de Photonique Biomédicale de l’Université d’Orsay, cet instrument sera mis à la disposition de la communauté scientifique.

Il s’agit d’un microscope de fluorescence à balayage où on superpose au faisceau d’excitation classiquement présent, un faisceau de déplétion mis en forme de doughnut afin de dépasser la limite de résolution imposée par la diffraction. Cette limite est dépassée en utilisant l'émission stimulée pour empecher l’émission de fluorescence dans la zone associée au doughnut. Ainsi seule la zone intérieure au doughnut peut encore émettre de la la fluorescence, et la largeur de cette zone centrale est ajustée grâce à la saturation des effets de déplétion de la fluorescence.

First images under the diffraction limit. Labelling establish for the biological applications

Improve lateral and axial resolution
Apply to Alzheimer disease investigation

Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging, PLOS One 2012,
Viviane Devauges, Catherine Marquer, Sandrine Lécart, Jack-Christophe Cossec, Marie-Claude Potier, Emmanuel Fort, Klaus Suhling, Sandrine Lévêque-Fort

The light microscope has been the key to many biological and medical discoveries. Due to its wave nature, light is subject to the phenomenon of diffraction, whose resolution-limiting effects were first described by Ernst Abbe in 1873. Structures which are closer to each other than ~ 250 nm cannot be visually separated when observed using visible light. Abbe's realization of the resolution limitation of the optical microscope was long thought to be an unalterable law of far-field light imaging. In recent years, S. Hell and J. Wichmann have been able to break the Abbe resolution limit of far-field optical microscopy, as applied to fluorescent imaging, using a technique known as Stimulated Emission Depletion (STED) microscopy. In conventional fluorescence microscopy, the excitation light excites fluorescent markers which tag molecules of interest in the sample. The markers are excited to a higher energy state, from which they emit light of a longer wavelength when they return to the ground state. By scanning this excitation spot over the sample and recording the emitted fluorescent light, one can form an image of the sample. The smaller the excitation spot is, the higher the resolution of the microscope. However, due to diffraction, the excitation spot cannot be made
smaller than ~ 250 nm. The trick with STED microscopy is that one uses a second beam (STED beam) to quench the fluorescent markers before they fluoresce. Because the STED beam is doughnut-shaped and centered over the excitation spot, one is able to preferentially quench the markers at the outer edge of the excitation spot and not those in the center. The result is a smaller effective fluorescence spot. By making the STED doughnut very intense, it is in principle possible to shrink the fluorescent spot to molecular size, thus attaining molecular resolution (20 nm was demonstrated recently) We propose to develop a STED microscope with the particularity to be tunable. The ability to change the optical wavelength of both the excitation and desexcitation (STED) beams is crucial in order to be able to excite the large variety of fluorophores commonly used for cell imaging. Ultrafast excitation will be exploited for fluorescence lifetime imaging using time-correlated single photon counting. Fluorescence nanoscopy combined with time-resolved imaging will provide a powerful tool for understanding of intracellular life processes, which may lead to revolutionary discoveries on the subject of how diseases originate. To avoid photobleching and facilitate live cells imaging, photophysical investigations of probes will be undertaken to optimzed STED beam parameters for different biological systems. During the project, we will focus on a neurobiological study related to Alzheimer disease mechanisms. We’ll localize APP (Amyloid precursor protein) and its clivage enzyme down to the synaptic vesicles (~50nm), their interaction using FRET/FLIM measurements, and displacements thanks to the real-time approach, at the neuronal axone level. Imaging of these processes has only been hampered by small area (below 100 nm) involved, which is below the resolution limit of conventional fluorescence microscopy. The original STED-FLIM microscope will be developed in the Biomedical Photonic Center of Orsay University, and will thus be open to the scientific community.

Project coordinator


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 450,000 euros
Beginning and duration of the scientific project: - 36 Months

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