Résumé : Stimulated Emission Depletion (STED) microscopy overcomes the diffraction barrier of conventional light microscopy, which limits resolution and thus useful magnification. STED microscopy allows using an increased magnification under physiological conditions.
I will demonstrate how simultaneous holographic photo-stimulation and super-resolution STED imaging can be achieved by incorporating Computer Generated Holography into a STED microscope. This system is applied to study the morphological changes of dendritic spines in neurons after stimulation.
Furthermore, Fast STED microscopy with 28 frames per second enabling the detailed analysis of the motion of synaptic vesicles in living neurons is shown. Dynamic imaging with up to 200 frames per second is exemplified by the observation of colloidal-crystal formation.
Whereas STED microscopy resolves nanostructures even in vivo, it is often difficult to relate super-resolved structures to other non-labeled features. Integrating phase contrast into a STED microscope provides a second, label-free contrast channel. This allows for easy correlation of morphological structures with high-resolution fluorescence images. It is demonstrated that Spiral Phase Contrast in scanning confocal configuration yields improved optical contrast and allows quantitative phase measurements. Scanning phase contrast allows for registration with the fluorescence images and for simultaneous recording of phase contrast and STED images. It enables therefore dual imaging and overlay in two contrast modes in fixed and in living specimen.
Lieu : LIPhy, conference room - 140 Avenue de la Physique 38402 Saint Martin d’Hères