Fig. 1: Confocal shadow imaging in organotypic brain slices.

a Schematic of imaging technique based on a commercial inverted confocal microscope equipped with a motorized correction collar to reduce optical aberrations. b Organotypic brain slices were placed (upside down) into an imaging chamber containing the membrane-impermeant organic dye diluted in ACSF (100 μM of Calcein, or 200 μM of Alexa Fluor 594). c Z-stack of confocal shadow images (Calcein, 100 μM) of CA1 area of hippocampus in an organotypic brain slice (see also SI Movies 1, 2 and 3; representative of 5 independent experiments). Scale bar, 20 μm. 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. d Time series of confocal shadow images (Calcein, 100 μM) of CA1 area of hippocampus in an organotypic brain slice, 1 frame every 5 s, 100 frames in total (see also SI Movie 4; representative series of 4 independent experiments). Scale bar, 20 μm. e Representative plot of line profiles across interstitial space between adjacent cell bodies (indicated by blue line) demonstrates no change in SNR across 100 consecutive frames (of 4 independent experiments).