Fig. 3: Light-sheet shadow imaging in combination with fast imaging of functional signals.

a Schematic of imaging technique based on a custom-built lattice light-sheet microscope. b Organotypic brain slices were placed (right side up) into an imaging chamber containing the membrane-impermeant organic dye diluted in ACSF (20 μM of Calcein). c Shadow images (representative of 6 independent experiments) acquired by light-sheet microscopy in different areas of the hippocampus in organotypic brain slices (see also SI Movies 8 and 9). Scale bars: 10 μm. d High-speed image z-stack (representative of 6 independent image acquisitions) in organotypic brain slice, 175 frames acquired at 80 Hz and a Δz step size of 200 nm. Scale bar, 5 μm. Left: Three examples of raw and Gaussian-fitted line profiles, color-coded with lines drawn on the image, indicating the spatial resolution in the tissue. FWHM: full-width half-maximum. e Schematic of two ways of visualization of 3D data acquired with the lattice light-sheet microscope: oblique light-sheet orientation or horizontal orientation, which is computationally generated by de-skewing, rotation, and image deconvolution. f Left: LISHI image viewed in the horizontal orientation taken from a z-stack. Scale bar: 10 μm; Right: Images (representative of 11 independent image acquisitions) of the same region in Alexa-568 and iGluSnFR channels, both in the light-sheet orientation. Numbered red boxes indicate the ROIs of glutamate transients in the light-sheet orientation and their corresponding locations (dotted red boxes) in the horizontal view. Scale bar: 5 μm. g Time traces of averaged fluorescence (iGluSnFR) in ROIs. h Left: LISHI images viewed in the horizontal orientation taken from a z-stack. Scale bar: 10 μm; Right: Images (representative of 3 independent image acquisitions) of the same region in Alexa-568 and GCaMP6f channels, both in the light-sheet orientation. Numbered red boxes indicate the ROIs of calcium transients in the light-sheet orientation and their corresponding locations (dotted red boxes) in the horizontal view. The dotted pink lines (i, ii) indicate the sections in the corresponding horizontal LISHI z stack (i, ii). Scale bar: 5 μm. i Time traces of averaged fluorescence (GCaMP6f) in ROIs.