Supplementary Figure 8: Crosstalk due to fluorescence scattering between adjacent image tiles is ≲8% at imaging depths ≲500 μm in tissue.

Assessments of how fluorescence scattering impacts the spatial distribution of fluorescence signals emitted by a localized source in tissue. A stationary laser beam induced localized two-photon excited fluorescence at different depths in fixed coronal brain slices from GCaMP6f-tTA-dCre mice expressing GCaMP6f in layer 2/3 neocortical pyramidal cells. The resulting fluorescence images captured by the sCMOS camera (Fig. 1a) allowed evaluations of the spatial distributions of fluorescence intensity as a function of the lateral distance from the laser focus. Data in this figure are based on averages of these distributions over all polar angles and 50 different individual locations of the laser beam for each of 51 different depths in tissue and 7 different cortical regions. These mean spatial distributions of fluorescence yielded the probability density functions in panels a–c, P_S (x, y), governing the lateral spatial distribution of detected fluorescence relative to a laser focal spot at x = y = 0, as well as the crosstalk probabilities in panel d, p_{i, j}, that a fluorescence photon excited by one beamlet in the laser illumination array (Fig. 1a) would scatter in tissue to such an extent that it could be detected on the camera in an image tile associated with a nearby laser beamlet, i tiles away in the x-dimension and j tiles away in the y-dimension. (a) Plots of P_S (x, y) for a laser beam positioned at the tissue surface (left) and 300 μm beneath the surface (right). The two square boxes within the right plot denote image tiles, each d = 24 μm wide, to the right of the laser focus for j = 0 and i = 1 and i = 2. Scale bars: 25 μm. (b) Left, Cross-sectional profiles of P_S (x, y), i.e., the functions P_S (x, 0), plotted for a range of different depths in tissue up to 500 μm beneath the tissue surface. Right, The same cross-sectional profiles but with each profile normalized such that P_S (0, 0) = 1. (c) 4 example cross-sectional profiles, P_S (x, 0), from panel b, illustrating how scattering affects the spatial distribution of fluorescence excited at 4 different depths in tissue. (d) Plots of the depth-dependent crosstalk probability (on a y-axis spanning 0-8%) that a fluorescence photon excited by one laser beamlet scatters to a nearby image tile corresponding to the location of either a nearest-neighbor, p_0,1, or a next-nearest neighbor beamlet, p_0,2, as computed from the measured P_S (x, y) distributions for both a 15-µm-separation (top) and a 24-µm-separation (bottom) between adjacent laser foci in the specimen plane. Note that with the larger, 24-µm-separation spacing between adjacent beamlets, the greater area per image tile counteracts the greater separation between tiles, such that the crosstalk probabilities are comparable to those for a 15-µm-separation between adjacent beamlets. These determinations of p_{i, j} informed subsequent image reconstructions based on sub-sampling (Supplementary Fig. 9). Shading denotes s.d. across n = 7 different locations in cortex. (e) Estimates of the depth-dependent decline in fluorescence Ca²⁺ signals that would occur in a neural Ca²⁺ imaging study, computed using the empirically determined P_S (x, y) distributions for 50 different depths in neocortical tissue. For each depth in tissue, the corresponding P_S (x, y) distribution was convolved with a uniform, circularly symmetric fluorescence source (12 µm in diameter) intended to model a neocortical neural cell body. Green curve shows the proportion of computed fluorescence signals remaining inside the cell body perimeter after this convolution. Black curve is a parametric fit to the signal decline as a function of the depth in tissue using a decaying exponential function, e^−z/z_S, with a characteristic decay length of z_S = 142 ± 30 µm (s.d.). Green shading denotes s.d. across the measurements taken at n = 7 different locations in cortex.