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High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations


Genetically encoded voltage indicators (GEVIs) hold immense potential for monitoring neuronal population activity. To date, best-in-class GEVIs rely on one-photon excitation. However, GEVI imaging of dense neuronal populations remains difficult because out-of-focus background fluorescence produces low contrast and excess noise when paired with conventional one-photon widefield imaging methods. To address this challenge, we developed an imaging system capable of efficient, high-contrast GEVI imaging at near-kHz rates and demonstrate it for in vivo and ex vivo imaging applications in the mouse neocortex. Our approach uses simultaneous multiplane imaging to monitor activity within contiguous tissue volumes with no penalty in speed or requirement for high excitation power. This approach, multi-Z imaging with confocal detection (MuZIC), permits high signal-to-noise ratio voltage imaging in densely labeled neuronal populations and is compatible with imaging through micro-optics. Moreover, it minimizes artifacts associated with concurrent imaging and optogenetic photostimulation for all-optical electrophysiology.

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Fig. 1: High-contrast multiplane imaging in densely labeled tissue with MuZIC.
Fig. 2: MuZIC enables high-SNR GEVI imaging in vitro.
Fig. 3: MuZIC reports subthreshold and suprathreshold activity in neural ensembles in vivo.
Fig. 4: Depth dependence of imaging with MuZIC.
Fig. 5: MuZIC can be performed through a microprism to visualize cells across cortical layers.
Fig. 6: Simultaneous imaging and optogenetic stimulation with minimal optical crosstalk.

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Data availability

Data described in this study are available on figshare ( Information describing construction of our MuZIC system is available via GitHub (detector:; assembly:

Code availability

All code used for data analyses is provided on figshare (


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We thank A. Abdelfattah, A. Singh, G. Murphy and K. Svoboda for technical advice and discussions. We thank E. Schreiter and I. Kolb for sharing Voltron2-ST reagents prepublication. We thank M. Giacomelli for advice on silicon photomultipliers. We thank T. Wang and D. Kleinfeld for comments on the paper. This work was supported by research grants from the National Institutes of Health: grant no. R01EB029171 (J.M.), grant no. RF1MH126882 (M.N.E.) and grant no. F32MH129149 (M.V.M.). The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations



T.D.W., M.V.M., J.M. and M.N.E. conceived of the project. M.N.E. and J.M. supervised research. T.D.W., J.M. and M.N.E. designed the MuZIC system. M.V.M., K.K. and M.N.E. prepared samples. T.D.W., M.V.M., K.K. and M.N.E. performed experiments. T.D.W., M.V.M. and M.N.E. analyzed data. T.D.W. performed simulations. T.D.W., M.V.M., J.M. and M.N.E. wrote the paper.

Corresponding author

Correspondence to Michael N. Economo.

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Competing interests

J.M., T.D.W. and Boston University have a US patent filed (no. 11,042,016) that relates to multiplane confocal imaging. The remaining authors declare no competing interests.

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Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Detailed system schematic.

The light source is a continuous-wave laser diode (‘561 nm’). Blue dashed box (‘Photo-stim.’) denotes components of the full-field photo-stimulation module. Green dashed box (‘Epi-illum.’) denotes components of the epi-illumination widefield fluorescence microscopy unit. M: mirror, Ir: iris, L: lens, DM: dichroic mirror, PS: polygonal scanner, GS: galvanometer scanner, SM: switchable mirror, OL: objective lens, FPs: focal planes, PH: pinhole, D: detector, C: camera. All part numbers are for the manufacturer Thorlabs, except the objective lens (Nikon).

Extended Data Fig. 2 System resolution.

Experimental measurements of the combined excitation and emission point spread functions (PSFs)corresponding to the four simultaneously acquired image planes. Confocal pinhole diameters were selected so that PSFs corresponding to adjacent detectors overlap, eliminating gaps between the four imaging planes, which together cover ~40 μm in the axial direction. Lateral resolution was somewhat anisotropic on account of the slower detector fall time. (X: 2.6 μm full-width half-max; Y: 1.9 μm).

Extended Data Fig. 3 Photobleaching.

a. Left, Example of raw, unprocessed fluorescence from all cells identified from the in vitro imaging experiment depicted in Fig. 6a,b. Right, Exponential fits to raw fluorescence normalized to initial brightness. These data were collected with the highest excitation power of all in vitro experiments (561 nm; 23.6 mW/mm2). b. Raw, unprocessed recorded from all cells identified in the in vivo imaging experiment depicted in Fig. 3b (top) and exponential fits to in vivo fluorescence, as in (a). The same excitation power was used for all in vivo imaging experiments (561 nm; 300 mW/mm2).

Extended Data Fig. 4 Multi-Z imaging minimizes axial motion artifacts.

a. During periods of brain motion, fluorescence is redistributed across planes, with cells becoming brighter in some planes and dimmer in others. Top, average change in fluorescence during a period of movement indicated by arrow below in adjacent planes. Bottom, average fluorescence time series corresponding to the cell indicated (yellow arrowhead). b. Top, mean fluorescence (left), difference image prior to (middle) and following (right)lateral motion correction. Sum of 4 image planes shown. During periods of brain motion (indicated by black arrow, bottom), lateral motion correction compensates for brain motion. Bottom, fluorescence time series corresponding to the cell indicated (yellow arrowhead, top). Time series extracted from a single plane(plane 2) before lateral motion correction (light blue, top), after lateral motion correction (dark blue, middle), and from a region of interest (ROI) spanning all four image planes following lateral motion correction (gray, bottom). Artifacts introduced by brain motion are suppressed when extracting time series from multiplane ROIs. Data are representative of axial motion across 34 fields, 5 animals.

Extended Data Fig. 5 Confocal collection efficiency.

Simulated confocal collection efficiency for fluorescence emitted from point sources (left) and 15-μm shells (right) located at different axial positions. Axial resolution is degraded as a result of spherical aberration induced by imaging through 0.5 mm cover glass (bottom; RI = 1.52). Lateral resolution is not degraded as much as axial resolution, meaning that the total fluorescence together collected by all detectors is only modestly reduced. See Methods and Supplementary Note 4 for more details.

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.

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Supplementary Notes 1–5 and Tables 1 and 2.

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Weber, T.D., Moya, M.V., Kılıç, K. et al. High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations. Nat Neurosci 26, 1642–1650 (2023).

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