Extended Data Fig. 4: Crosstalk un-mixing procedure for reconstructing the full field-of-view enables accurate estimation of neural activity traces.

a, To quantify the extent of fluorescence scattering across image tiles, we acquired images in two distinct configurations that enabled us to distinguish fluorescence signals from any crosstalk due to fluorescence scattering across image tiles. Using an awake mouse expressing GCaMP6f in layer 2/3 cortical pyramidal cells, we first imaged with only one active laser beam and its corresponding PMT; the other 15 beams were blocked (configuration 1). In this configuration, there is no fluorescence scattering into the active image tile from the other 15 tiles, only the signals from the active tile. In configuration 2, we blocked the beam that had previously been active, unblocked the other 15 beams, operated the microscope with the normal multiplexing approach, and again sampled signals from all 16 PMTs. To estimate the extent of scattering into the tile with the blocked beam, we applied the computational un-mixing procedure to the raw image data. To estimate how much scattered fluorescence affects cell sorting, we first extracted individual cells and their Ca²⁺ activity traces from the first dataset, attained in configuration 1 without crosstalk. We then summed the images, frame by frame, from the two datasets, to create a mock dataset comprising unscattered plus scattered fluorescence signals, from which we again computationally extracted cells and their activity traces. This enabled a direct comparison between two datasets containing the exact same patterns of neural activity, with and without fluorescence scattering from other image tiles. b, Activity traces for four example cells, enabling comparisons of the Ca²⁺ activity traces (top), ΔF(t)/F₀, and the resulting traces of the estimated spike counts (bottom), between the datasets with (red traces) and without (black traces) inter-tile scattering. The traces with and without inter-tile scattered fluorescence signals are nearly indistinguishable by eye. c, Histogram of the ratio of estimated spikes for the two datasets constructed in a, for all time bins (0.14 s per time bin) with an estimated spike count greater than 0.5. The mean ratio is 1.0 ± 0.06 (mean ± s.d.; N = 31 cells). Total number of time bins, 5,865. d–g, Studies of fluorescence scattering between the active image tiles in one temporal phase (Extended Data Fig. 2b) of the multiplexing scheme used for two-photon imaging. Throughout the paper, we corrected computationally for fluorescence scattering from active to inactive image tiles within each temporal phase of imaging (Extended Data Fig. 3c, Methods). This approach neglects the small amount of fluorescence scattering from active tiles to other active tiles, which in principle could also be computationally corrected using a more sophisticated method than the one we adopted. Hence, we examined experimentally the validity of our computational approach and the extent to which scattering between active tiles can be justifiably neglected. The amplitude of scattering between active tiles (d) varies with the location of each laser beam and its proximity to a tile boundary. We used fixed cortical tissue slices from adult GCaMP6f-tTA-dCre mice to measure the amplitude of such scattering effects when imaging at different depths within brain tissue. An image (e) of the spatial distribution of two-photon fluorescence excited 500 μm deep within a tissue slice shows that a majority of scattered fluorescence photons exits the brain tissue relatively near to the laser focus. By averaging over 100 different laser foci positions in each of 3 different brain slices, we determined the mean cross-sectional spatial profiles (f) of scattered fluorescence excited at different depths in tissue, as a function of the lateral displacement, x, from the laser focus. Profiles are shown normalized to unity at x = 0. The inset of f shows a magnified view of these cross-sectional profiles for x ∈ [–1,000 μm, –500 μm], that is, up to 1 mm away from the laser focus. We used these empirically determined scattering profiles to compute the probability (mean ± s.d.; N = 300 laser focus positions) (g) that a fluorescence photon originating in one active image tile would scatter into an adjacent active tile. Even when the laser focus is on the boundary of an image tile, this probability remains less than 0.02 for all tissue depths ≤ 600 μm. For our studies of layer 2/3 cortical pyramidal cells in live mice, the probability of a fluorescence photon scattering between active tiles is less than 0.01. In conclusion, computational corrections for fluorescence scattering that account solely for scattering from active to inactive tiles—and neglect scattering between different active tiles—are empirically well justified.