Lattice light sheet microscopy and photo-stimulation in brain slices

Lattice light sheet (LLS) fluorescence microscopy is a powerful recent technique for in vivo imaging of single and multicellular samples at very high spatio-temporal resolutions. We built a LLS microscope in which we added a photostimulation path to perform all-optical neurophysiological studies in rodent hippocampal brain slices. Thanks to the photo-stimulation path we could achieve fluorescence recovery after photobleaching (FRAP) or glutamate uncaging at spatially and temporally controlled regions of interest. Several fluorescence labelling protocols were employed depending on the imaged structure. Sub-micrometric neuronal elements such as spines or dendritic vesicles could be imaged down to ~20 μm below the surface. We demonstrate the performances of LLS in several ongoing studies: measurement of AMPA receptor surface diffusion at single spines, vesicular transport in dendrites, spontaneous and stimulated local calcium activity in neurons and astrocytes.

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