Abstract
Large scale simultaneous recording of fast patterns of neural activity remains challenging. Volumetric imaging modalities such as scanning-beam light-sheet microscopy (LSM) and wide-field light-field microscopy (WFLFM) fall short of the goal due to their complex calibration procedure, low spatial resolution, or high-photobleaching. Here, we demonstrate a hybrid light-sheet light-field microscopy (LSLFM) modality that yields high spatial resolution with simplified alignment of the imaging plane and the excitation plane. This new modality combines the selective excitation of light-sheet illumination with volumetric light-field imaging. This modality overcomes the current limitations of the scanning-beam LSM and WFLFM implementations. Compared with LSM, LSLFM captures volumetric data at a frame rate 50× lower than the rate of LSM and requires no dynamic calibration. Compared with WFLFM, LSLFM produces moderate improvements in spatial resolutions, 10 times improvement in the contrast when imaging fluorescent beads, and 3.2× the signal-to-noise ratio in the detection of neural activity when imaging live zebrafish expressing a genetically encoded calcium sensor.
Highlights
Volumetric fluorescence imaging is a powerful tool to uncover the patterns of neural activity that underlie brain function
To experimentally validate the light-sheet light-field microscopy (LSLFM)’s potential for high-resolution imaging, we imaged red fluorescent beads embedded in agarose with wide-field light-field microscopy (WFLFM), LSLFM, and light-sheet microscopy (LSM)
We instead imaged large objects (500 nm beads) and undersampled the image with ∼3× demagnification when quantifying the spot size of our imaging modalities. These design choices enabled simultaneous 10 Hz volumetric imaging with both LSM and light-field imaging. Whereas this volumetric imaging speed corresponded to a 10 Hz camera frame rate when using WFLFM and LSLFM, it corresponded to a 500 Hz camera frame rate when using LSM to image 50 layers per imaging volume
Summary
Volumetric fluorescence imaging is a powerful tool to uncover the patterns of neural activity that underlie brain function. The ability to simultaneously access hundreds to thousands of neurons enables sophisticated studies of the collective activity from individual or multiple neural populations [1,2]. Extracting such population-scale activity at cellular resolution is a key focus of optical technology development; such development seeks to increase both the scale and the resolution of neural recordings [3,4]. LSM attains large scale recordings by imaging the two-dimensional (2D) sheets over hundreds of micrometers in the planar direction of the sheet; the microscope extends from sheet imaging to volumetric imaging by scanning the sheet in the remaining third dimension.
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