Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators.

Peter Quicke,Herman V Jadan,Mark Neil,Pier L Dragotti,Pingfan Song,Simon R Schultz,Thomas Knöpfel,Carmel L Howe,Chenchen Song,Amanda J Foust

doi:10.1117/1.nph.7.3.035006

Peter Quicke, Herman V Jadan + Show 8 more

Open Access

https://doi.org/10.1117/1.nph.7.3.035006

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Abstract

.Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM’s volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity.Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions.Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM.Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM.Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM’s potential for studying dendritic integration and action potential propagation in three spatial dimensions.

Highlights

Cellular resolution voltage imaging enables direct observation of neuronal computation
We demonstrate that Light-field microscopy (LFM) can simultaneously image axially separated dendrites, enabling single-shot capture and localization of genetically encoded voltage indicators (GEVIs) fluorescence transients in the 3-D dendritic arbor
We show that LFM enables 3-D localization of dendritic and somatic GEVI fluorescence transients and compare the extent to which refocused and deconvolved light fields enable lateral and axial transient localization

Summary

Introduction

Cellular resolution voltage imaging enables direct observation of neuronal computation. Imaging neuronal processes with this technique require the imaged membranes to lie approximately flat in the microscope’s focal plane. As these experiments are typically performed in slices, the requirement for flat, healthy, and superficial cells represents a significant barrier Neurophotonics. Anselmi et al.[15] applied remote focusing to axially shift and tilt the wide-field focal plane as required by the sample, enabling calcium imaging along tilted dendrites. This adaptation, costs half of the fluorescence emission and is limited to a single tilted plane at a time. Point spread function (PSF) engineering via cubic phase masks[16] or spherical aberration[17] enables parallelized volumetric sample imaging when combined with lightsheet excitation; to our knowledge, these approaches have not successfully been implemented to image membrane voltage

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