Xiao, ShengCunningham, William J.Kondabolu, KrishnakanthLowet, EricMoya, Maria V.Mount, Rebecca A.Ravasio, CaraBortz, EmmaShaw, DanaEconomo, Michael N.Han, XueMertz, Jerome2025-05-052025-05-052024-06S. Xiao, W.J. Cunningham, K. Kondabolu, E. Lowet, M.V. Moya, R.A. Mount, C. Ravasio, E. Bortz, D. Shaw, M.N. Economo, X. Han, J. Mertz. 2024. "Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy." Nature Methods, Volume 21, Issue 6, pp.1094-1102. https://doi.org/10.1038/s41592-024-02275-w1548-70911548-7105https://hdl.handle.net/2144/50337Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.pp. 1094-1102Print-ElectronicenBiological sciencesTechnologyMedical and health sciencesDevelopmental biologyMicroscopy, confocalAnimalsMiceSignal-to-noise ratioArticle10.1038/s41592-024-02275-w0000-0001-8959-2388 (Xiao, Sheng)0000-0003-2310-1990 (Moya, Maria V)0000-0001-7442-8825 (Bortz, Emma)0000-0003-1828-9191 (Shaw, Dana)0000-0001-9893-1505 (Economo, Michael N)0000-0003-3896-4609 (Han, Xue)0000-0002-6343-9155 (Mertz, Jerome)964463