Miniaturized widefield microscope for high speed in vivo voltage imaging

Catherine A Saladrigas¹, Forest Speed², Alec Teel³, Mo Zohrabi¹, Eduardo J Miscles⁴, Gregory L Futia², Larry V Baker², Ye Zhang⁵, Ioannis Kymissis⁵, Victor M Bright⁴, Cristin G Welle^{6

7}, Diego Restrepo³, Juliet T Gopinath^{1

8

9}, Emily A Gibson²

Affiliations

¹ Department of Electrical, Energy and Computer Engineering, University of Colorado Boulder, CO 80309, USA.
² Department of Bioengineering, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
³ Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
⁴ Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
⁵ Department of Electrical Engineering, Columbia University, New York, NY 10027, USA.
⁶ Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, CO 80045, USA.
⁷ Department of Neurosurgery, University of Colorado Anschutz Medical Campus, CO 80045, USA.
⁸ Department of Physics, University of Colorado Boulder, CO 80309, USA.
⁹ Materials Science and Engineering Program, University of Colorado Boulder, Boulder, CO 80309, USA.

PMID: 41532107
PMCID: PMC12795426
DOI: 10.1364/BOE.576516

Miniaturized widefield microscope for high speed in vivo voltage imaging

Catherine A Saladrigas et al. Biomed Opt Express. 2025.

. 2025 Dec 2;17(1):1-11.

doi: 10.1364/BOE.576516. eCollection 2026 Jan 1.

Authors

Affiliations

¹ Department of Electrical, Energy and Computer Engineering, University of Colorado Boulder, CO 80309, USA.
² Department of Bioengineering, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
³ Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
⁴ Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
⁵ Department of Electrical Engineering, Columbia University, New York, NY 10027, USA.
⁶ Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, CO 80045, USA.
⁷ Department of Neurosurgery, University of Colorado Anschutz Medical Campus, CO 80045, USA.
⁸ Department of Physics, University of Colorado Boulder, CO 80309, USA.
⁹ Materials Science and Engineering Program, University of Colorado Boulder, Boulder, CO 80309, USA.

PMID: 41532107
PMCID: PMC12795426
DOI: 10.1364/BOE.576516

Abstract

Functional imaging in freely moving animals with genetically encoded voltage indicators (GEVIs) will open new capabilities for neuroscientists to study the behavioral relevance of neural activity with high spatial and temporal precision. However, miniaturization of an imaging system with sufficient collection efficiency to resolve the small changes in fluorescence yield from voltage spikes, as well as the development of efficient image sensors that are sufficiently fast to capture them, has proven challenging. We present a miniaturized microscope designed for voltage imaging, with a numerical aperture of 0.6, a 250 µm field of view, and a 1.3-1.6 mm working distance that weighs 16.4 g. We show it is capable of imaging in vivo voltage spikes from Voltron2 with a spike peak-to-noise ratio >3 at a framerate of 530 Hz.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig. 1.**
Overview of MiniVolt. (a) CAD schematic showing a cross-sectional view of MiniVolt. (b) Zemax optical design for the emission path of MiniVolt. (c) Image of a 50 micron fluorescent grid target taken with MiniVolt. (d) Photo of MiniVolt next to a penny for size reference. Optics in (a) and (b) correspond to following: $L_{1}$ - Edmund Optics 49-657, $L_{2}$ - Edmund Optics 49-656, $L_{3}$ - Edmund Optics 49-924, Emission filter- Chroma ET570lp (6 $\times$ 6 $\times$ 1 mm), and Dichroic- Chroma T550lpxr (14 $\times$ 14 $\times$ 1 mm). (e) Diagram of MiniVolt experimental setup.

**Fig. 2.**
Voltage recording with MiniVolt in a head-fixed mouse.(a) Average intensity projection (AIP) of NDNF interneurons expressing Voltron₂₅₅₂ in the visual cortex. Data was recorded at 530 frames per second (FPS) using an excitation intensity of 170 $mW / {mm}^{2}$ at the sample. (b) VolPy extracted spike template for region of interest (ROI) 3 marked in (a). (c) $Δ F / F$ time courses extracted from the 5 ROIs marked in (a). (d) Raw intensity time course for the data highlighted in (c). Baseline fluorescence (orange) was calculated using a lowpass filter at 1/3 Hz to remove the effects of photobleaching without risking the removal of low frequency subthreshold oscillations in the theta band (4-12 Hz). (e) The $Δ F / F$ time course was obtained by dividing the raw signal by the baseline. Prominent spikes (indicated by red dots) are identified using the template in (b) [6].

**Fig. 3.**
Example benchtop and MiniVolt voltage recordings. (a) AIP of a benchtop recording taken at 500 frames per second with 90 $mW / {mm}^{2}$ intensity. (b) $Δ F / F$ time courses for 6 ROIs indicated in (a). (c) AIP of a MiniVolt recording taken at 525 frames per second with 180 $mW / {mm}^{2}$ intensity. (d) $Δ F / F$ time courses for 6 ROIs indicated in (c). (e) Mean PNR for the time courses for ROIs shown in (b) and (d). (f) VolPy spike templates extracted from the benchtop recording (ROI1) and the MiniVolt recording (ROI1).

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Update of

Miniaturized widefield microscope for high speed in vivo voltage imaging.
Saladrigas CA, Speed F, Teel A, Zohrabi M, Miscles EJ, Futia GL, Baker LV, Zhang Y, Kymissis I, Bright VM, Welle CG, Restrepo D, Gopinath JT, Gibson EA. Saladrigas CA, et al. bioRxiv [Preprint]. 2025 Aug 6:2025.08.04.668551. doi: 10.1101/2025.08.04.668551. bioRxiv. 2025. Update in: Biomed Opt Express. 2025 Dec 02;17(1):1-11. doi: 10.1364/BOE.576516. PMID: 40799544 Free PMC article. Updated. Preprint.

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figshare/10.6084/m9.figshare.30486884

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R01 NS123665/NS/NINDS NIH HHS/United States

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Miniaturized widefield microscope for high speed in vivo voltage imaging

Affiliations

Miniaturized widefield microscope for high speed in vivo voltage imaging

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Update of

References

Associated data

Grants and funding

LinkOut - more resources

Full Text Sources