Extended Data Fig. 2: Simultaneous CASH and image-scan recording.
From: Fast optical recording of neuronal activity by three-dimensional custom-access serial holography
(a) Experimental design: A single GCaMP6f-expressing cell is targeted first with a cell-centered image scan (39×39 pixels, 38 ms scan time) followed by a two-shot CASH acquisition (50 μs) resulting in 26 Hz loop rate. (b) Example recording (130 s) of spontaneous activity showing (top to bottom) the motion of the animal, absolute fluorescence signal obtained from 5×5 CASH (green), imaging (black; pixel mean after image stack alignment), the brightest pixel of the aligned image stack (blue), the 2×5 CASH signal (green) and the imaging-derived neuropil signal (black; pixel mean across a concentric region around the cell body). (c) Top: Overlay of ΔF/F fluorescence traces after neuropil (NP) correction and normalization minimizing the global mean squared difference. The alignment yields 0.98 coefficient of correlation (cc). Middle: amplitudes of cell motion in x and y direction from alignment of image frames. Bottom: fluorescence images of the targeted cell at different instants as marked above (black lines). (d) Overlay of the single pixel trace before (blue) and after (black) x,y-alignment of the time lapse image sequence. (e) Overlay of a CASH (green) and imaging (black; same cell as above), but with 810 nm excitation for Ca2+-independent fluorescence (bottom), with x,y cell motion (middle) and running speed of the animal (top). (f) Top: overlay of single-trial CASH (green) and imaging (black) recordings (different cell) during presentation of a visual noise stimulus (gray bars) yielding 0.94 correlation. Middle: x,y cell motions. Bottom: Snapshot fluorescence images of the cell at instants indicated by bars above (black). (g) 2D representation of x,y cell motion during recordings in (b; left) and (f; right) with moving speed of the animal in false color. (h) Coefficient of correlation of GCaMP6f traces of simultaneous CASH and imaging recordings, analogous to (b) and (f), for all recorded cells (violet bars; median) and of the same traces after removal of photon noise by MLspike deconvolution fit (green bars; median). The number of observed cells are indicated above. Error bars represent SD.
