30 Nov A lattice light sheet microscope (LLSM) at BIC

A lattice light sheet microscope (LLSM) is fully operational and open to users at the BIC photonic. This microscope is a replica of the microscope developed by the team of Eric Betzig at HHMI Janelia Farms, USA ¹. LLSM allows very high imaging speed (up to 300 fr/s in 2D and 5 Hz in 3D) and spatial resolution with reduced phototoxicity. Contrary to conventional light sheet microscopes, high axial resolution (~ 0.5 µm) is maintained throughout the illuminated plane (20 µm long by 100 µm wide). Lateral resolution is diffraction limited ( ~ 250 nm). LLSM main application is dynamic imaging of small live samples ( ~ 1 mm³), mounted on 5 mm diameter cover slips. Our LLSM is equipped with a high power laser combiner with the following laser wavelengths (power): 405 nm (300mW), 488 nm (1W), 560 nm (2W), 642 nm (2W). Furthermore, we added a photostimulation module to optically target any user-defined ROI with high spatiotemporal resolution. We validated this tool for FRAP of single spines or dendrites or glutamate uncaging. Photostimualtion can be achieved at any wavelength of the laser combiner (405, 488, 560 or 642 nm).

A. The lattice light sheet microscope setup. LC: laser combiner; BS: beam shaper composed of a spatial light modulator (SLM) and an annular mask (AM) to create the non-diffractive lattice pattern; SU: scanning unit with Z and X galvos (GZ and GX) translates the pattern along the x and z axis; EO excitation objective, DO detection objective; Inset: the light sheet is a very thin (~ 0.5 µm) and extended plane (>15 µm along Y and >100 µm along X). PSM: photostimulation module. L: any laser line from the combiner. XYT: galvo-based targeting system. B. 3D imaging of vesicular transport in neuronal dendrites 4.4 Vol/s, C. Targeted photobleaching can be used to increase contrast of vesicles in dendrite (10 fr/s, total time 3mn). D. Spontaneous calcium activity in an astrocyte expressing GCamp6 protein imaged at 0.9 Vol/s (B and C courtesy of Valeria Pecoraro, IINS, Choquet Team, D: Labelling done by Monica Fernandez Monreal ).