Figure 2. Quantification of TICO microscope performance for in vivo voltage imaging.

(a-c) Example Voltron2 fluorescence images under targeted illumination with confocal slit width set to 4.5, 22.5, and 156 µm. Scale bar 50 µm.

(d) Voltron2 fluorescence image over the same FOV but acquired without targeted illumination and with a confocal slit width of 156 µm. TI, targeted illumination. Scale bar 50 µm.

(e,h,i) Comparison of spike $\Delta F/F$, spike detection fidelity $d\prime$, and spike SNR measured with targeted illumination and confocal slit widths of 4.5, 11.3, 22.5, and 156 µm (n = 30 cells from 6 FOVs, 2 mice). Box plots: box, 25th (Q1, bottom line) to 75^th (Q3, top line) percentiles; whiskers, $Q1-1.5\times IQR$ to $Q3+1.5\times IQR$, where $IQR=Q3-Q1$; middle line, median (m); notch, from $m-1.57\times IQR/\sqrt{n}$ to $m+1.57\times IQR/\sqrt{n}$; dots, measurement points. p < 0.05, p < 0.01, p < 0.001, no label if p ≥ 0.05, pairwise Wilcoxon signed-rank test, see Table S4 for statistics.

(f,g,j) Comparison of spike $\Delta F/F$, photobleaching rate, and spike SNR measured with and without targeted illumination when using a 14 µm confocal slit. For (f,j), n = 19 cells from 5 FOVs, 2 mice. For (g), n = 92 cells from 5 FOVs, 2 mice.

(l,m,n) Example images (scale bar, 20 µm) and corresponding fluorescence traces from two neighboring neurons with targeted illumination and confocal slit widths of 4.5, 22.5, and 156 µm (from top to bottom). Gray line, fluorescence traces; red line, extracted subthreshold Vm traces; r, Pearson cross-correlation coefficient between the subthreshold Vm traces from the 2 neurons.