Figure 5

Comparisons in detected ROIs and their activity between 8K-SDCLM and TPLSM imaging. (a) Representative distribution of the normalized fluorescence signals from one pixel in the field shown in Fig. 4ai (21,600 time points). The fitted gamma distribution is also shown (the shape parameter is 2.35 and scale parameter is 2.87). (b) Relationship between the variance and time-averaged normalized fluorescence signal for 100 pixels. The red cross indicates the pixel shown in a. The regression line is also shown (p = 1.81 × 10⁻⁷⁵, Pearson correlation test). (c) Representative 30 frame-averaged TPLSM images with the artificial noise (from left to right, NR = 0, 1, 2, 3, 3.71, and 5). The ROI area corresponding to the left-most one was extracted from each image and its contour was overlaid on each image. Scale bar, 5 µm. (d) ΔF/F traces of the ROI areas shown in c. Black traces were directly calculated from the fluorescence signals from the motion-corrected raw images with artificial noise. Orange traces were denoised traces obtained using the CNMF algorithm. (e) Ratios of the number of detected ROIs (black), the number of detected spike events (red), and the pair-wise correlation coefficients in the spike event between pairs of ROIs (blue) with and without the artificial noise are plotted against the added noise (NR = 1, 2, 3, 3.71, and 5). The dotted line indicates the NR of 3.71. (f) Cumulative distribution of the ROI area in all ROIs without the noise (gray, n = 1697) and the robust ROIs (black, n = 278). p = 0.797, Kolmogorov-Smirnov test. (g) Cumulative distribution of F in all ROIs without the noise (gray, n = 1697) and the robust ROIs (black, n = 278), p = 0.402, Kolmogorov-Smirnov test. (h) Cumulative distribution of the maximal amplitude of ΔF/F in all ROIs without the noise (gray, n = 1697) and the robust ROIs (black, n = 278). p = 1.84 × 10⁻⁵⁷, Kolmogorov-Smirnov test. (i) Schematic illustration of XYZ TPLSM imaging of TC axons at three depths. (j) Representative spatial distribution of ROIs extracted from three 8 µm-apart fields in the same horizontal location. The left panel is the composite image of the ROIs from planes 1, (blue), 2 (orange), and 3 (green). Black areas are overlapping areas of pairs of ROIs. In the right panel, the overlapping areas of pairs of ROIs in the left panel are colored (cyan or magenta), while the ROI areas with no overlap between different planes in the left panel are colored gray. Arrowheads indicate pairs of ROIs whose overlapping area exceeded 50% of the ROI area. Magenta arrowheads indicate overlapping areas of pairs of ROIs with a pair-wise correlation coefficient in spike events of >0.6, which were removed for the calculation of the overlapping ratio (see Methods for details). Scale bar, 10 µm.