Miniaturized widefield microscope for high speed in vivo voltage imaging

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 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, 250 μm field of view and 1.3 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.

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 fluorescent grid target taken with MiniVolt. Scale bar corresponds to 50 microns. (d) Photo of MiniVolt next to a penny for size reference. Optics in (a) and (b) correspond to following: L₁- Edmund Optics 49–657, L₂- Edmund Optics 49–656, L₃- Edmund Optics 49–924, Emission filter- Chroma ET570lp (6×6×1 mm), and Dichroic- Chroma T550lpxr (14×14×1 mm)

Fig. 2.

(a) Average intensity projection (AIP) of a 500 Hz MiniVolt recording of NDNF-Cre expressing cells in the visual cortex. (b) Raw intensity time course extracted using MiniVolt from the region of information marked in (a). 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). ΔF/F time course (bottom) was obtained by dividing the raw signal by the baseline. Prominent spikes (indicated by red dots) are identified using the template in (c). (c) VolPy extracted spike template used to identify prominent spikes [6]

Fig. 3.

Example voltage recording using MiniVolt in a head-fixed mouse. (a) AIP of NDNF interneurons expressing Voltron2₅₅₂ in the visual cortex. Data was recorded at 530 Hz with 170 mW/mm² intensity at the sample. (b) ΔF/F time courses for 5 regions of information indicated in (a). (c) Zoomed-in time course for the yellow highlighted region in (b). The red points above the time course are the timing of spiking events identified using VolPy

Fig. 4.

Example benchtop and MiniVolt voltage recordings. (a) AIP of a benchtop recording taken at 500 frames per second with 90 mW/mm² intensity. (b) ΔF/F time courses for 6 ROIs indicated in (a). (c) AIP of a MiniVolt recording taken at 525 frames per second with 180 mW/mm² intensity. (d) ΔF/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).