Light-field microscopy for fast volumetric brain imaging
Introduction
The central nervous system of animals consists of a large number of neurons, which vary in form and function and have complex connections among them. When studying complex behaviors, recording the activity of a single neuron or a small number of neurons is usually insufficient to obtain a complete picture of the information processing flow. Therefore, simultaneous recording of neural activity over large neuronal populations is considered one of the keys for better understanding of how high-order functions are achieved in such complex brains (Yang and Yuste, 2017).
Over the past 20 years, chemical dyes and genetically encoded fluorescent proteins that can indicate neuronal activity have been introduced and optimized gradually in an effort to achieve high-throughput optical readout of neuronal activity at single neuron resolution. Among them, the GCaMP series of calcium indicators have become the most widely applied indicators (Chen et al., 2013; Dana et al., 2019; O’Banion and Yasuda, 2020). Their combination with a new type of microscope, the two-photon laser scanning fluorescence microscope (Denk et al., 1990), which can penetrate deep into scattering tissues, has made it possible to record neural population activity in living brains (Stosiek et al., 2003; Wang et al., 2003). Within a single field of view, hundreds of neurons with their positional, morphological, and genetic specificity can usually be recorded at the same time using optical imaging strategies, albeit with lower temporal resolution than that in conventional electrophysiology techniques. Furthermore, the high spatial resolution and non-invasive imaging method afforded by optical microscopes makes it possible to record calcium signals in dendrites, axons, and even individual dendritic spines (Svoboda et al., 1997; Wachowiak et al., 2004; Wang et al., 2003; Yuste and Denk, 1995), which has substantially transformed the methods of functional studies in neurosciences.
In addition to the ever-growing number of calcium indicators, probes for neurotransmitters and other important chemicals in the brain, which are not detectable using conventional electrophysiology, have also been developed (Jing et al., 2018; Marvin et al., 2018; Sun et al., 2018). In particular, genetically encoded voltage indicators, which could overcome the limitations on the temporal resolution of calcium imaging, have been progressively optimized (Peterka et al., 2011). Several recent studies have demonstrated that these genetically encoded voltage indicators can report action potentials and subthreshold membrane potentials of neurons in the brains of behaving mice and other model organisms (Abdelfattah et al., 2019; Adam et al., 2019; Piatkevich et al., 2019; Villette et al., 2019). Moreover, functional optical imaging can be readily combined with optogenetic manipulation to achieve optical electrophysiology at high resolution and throughput (Fan et al., 2020; Hochbaum et al., 2014). These research tools can be further optimized and promise important directions for future technology development in neuroscience studies.
Along with rapid developments in fluorescent indicators, optical imaging techniques are continuously evolving to cope with increasingly challenging tasks in neuroscience studies. Traditional two-photon microscopy uses a tightly focused laser spot that ensures efficient nonlinear fluorescence excitation to scan a plane or volume in a point-by-point manner (Fig. 1A). This serial information collection scheme is often limited in speed by the achievable scanning speed of the galvo mirrors and piezo stages. To overcome this problem, various new strategies have been developed to either make use of faster scanning mechanisms or explore the spatiotemporal sparsity of the neural signal to collect information in compressed forms (Grewe et al., 2011; Han et al., 2019; Katona et al., 2012; Nikolenko et al., 2008; Sofroniew et al., 2016; Weisenburger et al., 2019; Yang et al., 2016; Zhang et al., 2019). However, the fluorescence saturation, animals’ tolerance of laser powers, and the signal density still set limits for the above-mentioned approaches. When the tissue is not heavily scattering and imaging depth is not demanding, one-photon excitation-based wide-field fluorescence imaging strategies allow greatly increased imaging throughput by parallelizing information collection using cameras. For example, wide-field detection using selective fluorescence excitation localized near the focal plane forms a light-sheet microscope that can image a 2D plane at a time and achieve a much higher imaging speed. However, it still requires scanning in the third direction to cover the volume (Fig. 1B). If conventional whole field epi-illumination is employed, the fluorescence in the entire 3D volume will be excited at the same time (Fig. 1C); however, the conventional wide-field detection scheme cannot discriminate signals from different depths and therefore lacks volumetric imaging capabilities (Fig. 2A). In contrast, light field microscopy (LFM) solves this problem elegantly by recording both the direction and location of light rays and achieving scanning-free and instantaneous volumetric imaging with a single camera exposure (Fig. 2B). In this way, the volumetric imaging speed in LFM is only limited by the camera’s fastest frame rate, which can typically reach several hundred hertz in modern CMOS cameras. However, the increased speed and volume coverage leads to a reduction in spatial resolution, and this trade-off can be flexibly tuned to achieve optimal performance in different applications. In addition, LFM has the advantages of a simple optical design and low cost, making it ideal for different laboratories to optimize for their own needs. For example, when integrated neural signals in somas, instead of those in dendrites or spines, are of interest, LFM can be optimized to perform calcium imaging at single-neuron resolution with considerably higher throughput than conventional two-photon scanning microscopes can typically achieve. Additionally, the completely parallelized imaging process alleviates the peak excitation laser power, providing the additional advantages of low photobleaching and photodamage (Levoy et al., 2006).
Section snippets
Principles and development of light-field microscopy
The original concept of LFM was developed in the field of photography. In 1908, Gabriel Lippmann conceived the concept of “integral photography”, aiming to use a lens array to modulate light so that one photograph could determine the appearance of an object in the original three-dimensional space, which was the prototype of the light-field camera. Approximately a century later, with the development of digital image sensors and digital image processing capability aided by computers, light-field
Light-field imaging of Caenorhabditis elegans, Drosophila, and zebrafish larvae
Understanding the integration of sensory input, motor output and feedback, and flow and processing of information among various brain regions at the whole-brain scale is one of the greatest challenges in neuroscientific studies (Alivisatos et al., 2012; Freeman et al., 2014) and requires tools to capture neural activities across the entire brain. On a macroscale, whole brains in rats, monkeys, and even humans can be imaged using functional magnetic resonance imaging (fMRI) (Weber et al., 2006).
LFM for the rodent brain
Two-photon microscopy, instead of wide-field imaging techniques, is the most widely applied method for functional imaging of neural activities in rodent brains because of its exceptional penetration depth and signal detection sensitivity in scattering tissues. However, its serial imaging scheme fundamentally limits its throughput and scalability to cover large volumes at high speed. Therefore, the application of LFMs in larger brains to gain a balance between penetration depth and imaging
Discussion
LFM has emerged as a promising imaging technique to capture extremely fast volumetric dynamics. One important direction that will soon become widely applied is the imaging of membrane potentials by combining its use with state-of-the-art voltage indicators. Very recently, various voltage indicators have been demonstrated in in vivo applications to monitor action potentials and subthreshold activities (Abdelfattah et al., 2019; Adam et al., 2019; Piatkevich et al., 2019; Villette et al., 2019).
CRediT authorship contribution statement
Zhenkun Zhang: Conceptualization, Writing - original draft, Visualization. Lin Cong: Writing - review & editing. Lu Bai: Writing - review & editing. Kai Wang: Conceptualization, Writing - original draft, Writing - review & editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
K.W. acknowledges supports from National Key R&D Program of China (2017YFA0700500), Strategic Priority Research Program of the Chinese Academy of Sciences (XDB32030200), International Partnership Program of Chinese Academy of Sciences (153D31KYSB20170059), Shanghai Municipal Science and Technology Major Project (2018SHZDZX05), NSFC (31871086) and China Thousand Talents Program.
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