Imaging the voltage of neurons distributed across entire brains of larval zebrafish

Abstract

Neurons interact in networks distributed throughout the brain. Although much effort has focused on whole-brain calcium imaging, recent advances in genetically encoded voltage indicators (GEVIs) raise the possibility of imaging voltage of neurons distributed across brains. To achieve this, a microscope must image at high volumetric rate and signal-to-noise ratio. We present a remote scanning light-sheet microscope capable of imaging GEVI-expressing neurons distributed throughout entire brains of larval zebrafish at a volumetric rate of 200.8 Hz. We measured voltage of ∼1/3 of the neurons of the brain, distributed throughout. We observed that neurons firing at different times during a sequence were located at different brain locations, for sequences elicited by a visual stimulus, which mapped onto locations throughout the optic tectum, as well as during stimulus-independent bursts, which mapped onto locations in the cerebellum and medulla. Whole-brain voltage imaging may open up frontiers in the fundamental operation of neural systems.

Scanless two-photon voltage imaging

“…However, the effective FOV used for individual high-speed voltage imaging recordings was reduced in one dimension due to the maximum number of rows of pixels that could be read out at a given acquisition rate. An alternative approach would be to replace the sCMOS sensor with a detector capable of higher (full frame) readout speeds such as single-photon avalanche diode (SPAD) arrays 95 , or multiple cameras 96 . The FOV of scanless 2P voltage imaging could also be trivially increased by reducing the magnification of the detection axis.…”

Two-photon voltage imaging with rhodopsin-based sensors

“…However, to date, only the ASAP-type sensors have been successfully used for AP detection under 2P illumination [7][8][9][10][11][12] . Although rhodopsin-based sensors are intrinsically brighter than the ASAP-type ones 6,13 , they are considered incompatible with 2P illumination and no 2P detection of APs has been reported to date 14,15 .…”

Kilohertz volumetric imaging of in-vivo dynamics using squeezed light field microscopy

“…The integration of ultra-high-speed cameras 7,8,21 and event cameras 22 holds promise for providing higher bandwidths to LFM. However, their current limitations in sensitivity and noise performance present challenges, especially for photon-starved applications like imaging genetically encoded voltage indicators (GEVIs).…”

“…Traditional three-dimensional (3D) imaging tools, such as confocal microscopy, light sheet microscopy, and two-photon microscopy, heavily rely on scanning to acquire a volumetric image. Despite advancement in beam shaping 5,6 , remote refocusing mechanisms 7 and detection geometry 8 , there persists an inherent trade-off between temporal resolution, the 3D field-of-view (FOV), and spatial resolution in these techniques. This constraint marks a significant challenge to obtain optimal performance across a large 3D field of view (FOV) for robust ultrafast detection exceeding kilohertz (kHz).…”

“…However, their current limitations in sensitivity and noise performance present challenges, especially for photon-starved applications like imaging genetically encoded voltage indicators (GEVIs). 7 On the other hand, the compressibility of fourdimensional (4D) (two spatial dimensions plus two angular dimensions) light fields has been leveraged for compressive detection. Coded masks [23][24][25] and diffusers 26 are employed to modulate and integrate the spatio-angular components originally recorded by distinct pixels.…”