Fig. 5: Mesoscale, cellular-resolution Ca²⁺ imaging using SeeThrough.

a Schematic of the integration of SeeThrough and FASHIO-2PM. b A camera image. The yellow square indicates the field-of-view (FOV). Overlaid white lines show borders according to the Allen mouse common coordinate framework. c A standard deviation projection image for Ca²⁺ imaging of a contiguous full FOV including layer 2 cortical neurons labeled with G-CaMP7.09 at 7.65 Hz sampling rate (150 μm depth from the cortical surface). Representative neurons are shown around the image. d An overlaid diagram showing 2965 regions of interest (ROIs). The colors correspond to the Ca²⁺ signals shown in (e). e Representative Ca²⁺ signals and deconvolved signals from 14 neurons. f Z-scored deconvolved signals of individual neurons recorded using FASHIO-2PM across 13 cortical regions in the same representative mouse. Neurons are sorted by region, and the global mean activity of all neurons is plotted above the plot. The color bar indicates the corresponding cortical regions. g Partial correlation coefficient matrix quantifies the pairwise relationships between the activities of individual neurons in the representative mouse, after removing the influence of the global mean activity. h Distribution of partial correlation coefficients for all neuron pairs in the representative mouse. i Distribution of partial correlation coefficients for individual mice. The inner lines indicate the quartile ranges (25th–75th percentiles) and the median. j Mean partial correlation coefficients calculated in 100 µm bins as a function of distance, averaged across individuals (n = 3 mice). Error bars represent the SEM. k Binary adjacency matrix for the same mouse as in (g), obtained by thresholding the partial correlation coefficients at the 99th percentile (partial correlation >0.184). l Visualization of 2000 randomly selected links from (k), color-coded by distance. Links shorter than 500 µm are shown in cyan, those between 500–1500 µm in magenta, and links longer than 1500 µm in yellow. m Degree distribution of neurons for the same mouse in (k). n Visualization of links for high-degree neurons. Links are color-coded based on degree rank: cyan for neurons in the top 5–2.5%, magenta for the top 2.5–1%, and yellow for the top 1%.