Volumetric Imaging of Neural Activity by Light Field Microscopy

Abstract

Recording the highly diverse and dynamic activities in large populations of neurons in behaving animals is crucial for a better understanding of how the brain works. To meet this challenge, extensive efforts have been devoted to developing functional fluorescent indicators and optical imaging techniques to optically monitor neural activity. Indeed, optical imaging potentially has extremely high throughput due to its non-invasive access to large brain regions and capability to sample neurons at high density, but the readout speed, such as the scanning speed in two-photon scanning microscopy, is often limited by various practical considerations. Among different imaging methods, light field microscopy features a highly parallelized 3D fluorescence imaging scheme and therefore promises a novel and faster strategy for functional imaging of neural activity. Here, we briefly review the working principles of various types of light field microscopes and their recent developments and applications in neuroscience studies. We also discuss strategies and considerations of optimizing light field microscopy for different experimental purposes, with illustrative examples in imaging zebrafish and mouse brains.

Keywords: Brain activity; Calcium imaging; Light field microscopy; Voltage imaging; Volumetric imaging.

Fig. 1

Principles of light field microscopy. A Schematics of wide-field microscopy, which forms in-focus (green) and out-of-focus (orange and blue) images simultaneously on the image sensor. B Schematics of conventional light field microscopy, which places a lenslet array at the focal plane and moves the image sensor back to the focal plane of the lenslet array. It does not form an image in the conventional sense but captures both positional and directional information of light rays using an image sensor. For example, the positional and directional information of Ray 1 is measured by its intersection with microlens L1 and camera pixel P1. This information is combined computationally to reconstruct a 3D light field, which can then be deconvolved to yield a 3D image, shown as dashed circles. C Schematics of Fourier light field microscopy, which places the lenslet array at the pupil plane of the objective by inserting a relay lens after the tube lens. The image sensor captures projection views from each lenslet. These projection views are combined computationally to reconstruct a 3D image. D The lenslet array is placed at the pupil plane of the objective in Fourier light field microscopy and each lenslet collects light from a limited NA_lenslet. The DOF of light field microscopy is estimated as the Rayleigh range of a beam with diverging angle specified by NA_lenslet. E The in-plane resolution of Fourier light field microscopy depends on the NA of each lenslet. The axial resolution depends on both the NA_lenslet and the NA_Obj of the imaging objective used, where NA_Obj is the NA of the imaging objective. F The DOF, or the axial coverage, decreases as NA_lenslet increases.

Fig. 2

Applications of light field microscopy in neuroscience. A Imaging neuronal activity in small animals by conventional light field microscopy. Left, schematics of traditional light field microscopy (scale bar, 150 μm). Middle and right, example images of C. elegans and its neuronal activity traces captured by light field microscopy (scale bar, 10 μm) (adapted with permission from Springer Nature [20]). B Light field imaging of freely-swimming larval zebrafish. Left, schematics of extended field-of-view light field microscopy. Middle, whole-brain functional imaging during prey capture behavior of larval zebrafish. Right, quantification of larval zebrafish behavior and neuronal dynamics during a successful prey capture of a paramecium (adapted with permission from Cong et al., 2017 [17]). C Volumetric imaging of the mouse brain by confocal light field microscopy. Left, concept of generalized confocal detection. Middle, confocal light field microscopy captures most of the neurons within ~400 μm deep into the mouse cortex, as confirmed by two-photon microscopy. Right, confocal light field microscopy captures >50,000 volumes without causing significant photobleaching (adapted with permission from Springer Nature [24]). D Voltage imaging of dendritic branches in acute mouse brain slices by light field microscopy. Left, schematic of light field microscopy optimized in resolution and imaging speed for voltage imaging; Middle, example regions of interests (ROIs) in light field imaging; Right, extracted voltage signals in these ROIs. sCMOS, scientific complementary metal-oxide-semiconductor; LFM, light field microscopy; MLA, micro-lens array; W.F., wide field; Decon., deconvolved; Refoc., refocused (adapted with permission from Quicke et al., 2020 [45]).