Extended Data Fig. 6: Comparison of MuZIC and widefield fluorescence microscopy (WFM).

a. Signal-to-noise ratio of MuZIC relative to WFM increases with increasing WFM background-to-signal ratio (BSR). SNR of MuZIC is mostly insensitive to out-of-focus background fluorescence, while SNR of WFM decreases with increasing background. When BSR is greater than about 5, SNR of MuZIC is higher than SNR of WFM (assuming contrast of MuZIC is 0.6 and detection efficiency of MuZIC is 25% relative to WFM; see Supplementary Note 2). b. WFM image of Voltron2-labeled neurons in vivo. Representative BSRs associated with different neurons are indicated for reference. Individual neurons were difficult to resolve in regions of higher labeling density (representative of 4 animals). c. Example in-vivo widefield images of neurons without (top) and with (bottom) targeted illumination delimited by dashed circles. Dashed circles span regions of interest encompassing a single neuron (top left: signal + background) and background only (top right). Corresponding images when targeted illumination was applied (bottom) are shown recentered. In this extreme example where only a single neuron is targeted, the BSR is found to be reduced by a factorγ = 0.2(see Supplementary Note 3, representative of 4 animals). d. Signal-to-noise ratio of MuZIC relative to WFM without (solid) and with (dashed) the addition of targeted illumination of a single neuron (that isγ= 0.2). In general, when multiple neurons are targeted, the reduction in BSR is not as significant and the relative SNR gain provided by MuZIC lies within the shaded region. Targeted illumination has the effect of shifting the break-even point where MuZIC becomes advantageous over WFM to a higher BSR value, up to about 25 in the very extreme case of single-neuron targeted illumination.