Light-field microscopy for fast volumetric brain imaging

https://doi.org/10.1016/j.jneumeth.2021.109083Get rights and content

Highlights

  • Light field microscopy is a fast volumetric imaging technique.

  • Light field microscopy can capture very fast dynamics, such as neural activities and blood flows.

  • Light field microscopy can capture whole brain neural activity in freely moving zebrafish.

  • Light field microscopy can be improved by introducing optical sectioning capability.

Abstract

Recording neural activities over large populations is critical for a better understanding of the functional mechanisms of animal brains. Traditional optical imaging technologies for in vivo neural activity recording are usually limited in throughput and cannot cover a large imaging volume at high speed. Light-field microscopy features a highly parallelized imaging collection mechanism and can simultaneously record optical signals from different depths. Therefore, it can potentially increase the imaging throughput substantially. Furthermore, its unique instantaneous volumetric imaging capability enables the capture of highly dynamic processes, such as recording whole-animal neural activities in freely moving Caenorhabditis elegans and whole-brain neural activity in freely swimming larval zebrafish during prey capture. Here, we summarize the principles of and considerations in the practical implementation of light-field microscopy as currently applied in biological imaging experiments. We also discuss the strategies that light-field microscopy can employ when imaging thick tissues in the presence of scattering and background interference. Finally, we present a few examples of applying light-field microscopy in neuroscientific studies in several important animal models.

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.

References (83)

  • W. Yang et al.

    NeuroResource simultaneous multi-plane imaging of neural circuits

    Neuron

    (2016)
  • Y. Ziv et al.

    Miniature microscopes for large-scale imaging of neuronal activity in freely behaving rodents

    Curr. Opin. Neurobiol.

    (2015)
  • A.S. Abdelfattah et al.

    Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

    Science (80-.)

    (2019)
  • Y. Adam et al.

    Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics

    Nature

    (2019)
  • E.H. Adelson et al.

    The Plenoptic Function and the Elements of Early Vision

    (1991)
  • E.H. Adelson et al.

    Single Lens stereo with a plenoptic camera

    IEEE Trans. Pattern Anal. Mach. Intell.

    (1992)
  • M.B. Ahrens et al.

    Whole-brain functional imaging at cellular resolution using light-sheet microscopy

    Nat. Methods

    (2013)
  • S. Aimon et al.

    Fast near-whole–brain imaging in adult Drosophila during responses to stimuli and behavior

    PLoS Biol.

    (2019)
  • A.S. Andalman et al.

    Neuronal dynamics regulating brain and behavioral state transitions

    Cell

    (2019)
  • K. Bhattacharyya et al.

    Visual threat assessment and reticulospinal encoding of calibrated responses in larval zebrafish

    Curr. Biol.

    (2017)
  • M. Broxton et al.

    Wave optics theory and 3-D deconvolution for the light field microscope

    Opt. Express

    (2013)
  • J.A. Calarco et al.

    Imaging whole nervous systems : insights into behavior from worms to fish

    Nat. Methods

    (2019)
  • T.W. Chen et al.

    Ultrasensitive fluorescent proteins for imaging neuronal activity

    Nature

    (2013)
  • Y. Chen et al.

    Design of a high-resolution light field miniscope for volumetric imaging in scattering tissue

    Biomed. Opt. Express

    (2020)
  • N. Cohen et al.

    Enhancing the performance of the light field microscope using wavefront coding

    Opt. Express

    (2014)
  • L. Cong et al.

    Rapid whole brain imaging of neural activity in freely behaving larval zebrafish (Danio rerio)

    Elife

    (2017)
  • H. Dana et al.

    High-performance calcium sensors for imaging activity in neuronal populations and microcompartments

    Nat. Methods

    (2019)
  • W. Denk et al.

    Two-photon laser scanning fluorescence microscopy

    Science (80-.)

    (1990)
  • L.Z. Fan et al.

    All-optical electrophysiology reveals the role of lateral inhibition in sensory processing in cortical layer 1

    Cell

    (2020)
  • B.A. Flusberg et al.

    High-speed, miniaturized fluorescence microscopy in freely moving mice

    Nat. Methods

    (2008)
  • J. Freeman et al.

    Mapping brain activity at scale with cluster computing

    Nat. Methods

    (2014)
  • K.K. Ghosh et al.

    Miniaturized integration of a fluorescence microscope

    Nat. Methods

    (2011)
  • B.F. Grewe et al.

    Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens

    Biomed. Opt. Express

    (2011)
  • L. Grosenick et al.

    Identification of cellular-activity dynamics across large tissue volumes in the mammalian brain

    bioRxiv

    (2017)
  • C. Guo et al.

    Fourier light-field microscopy

    Opt. Express

    (2019)
  • S. Han et al.

    Two-color volumetric imaging of neuronal activity of cortical columns

    Cell Rep.

    (2019)
  • J.W. Hardy

    Adaptive optics for astronomical telescopes

    Oxford University Press on Demand.

    (1998)
  • D.R. Hochbaum et al.

    All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

    Nat. Methods

    (2014)
  • F.-C. Hsu et al.

    Volumetric bioimaging based on light field microscopy with temporal focusing illumination

    Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV

    (2018)
  • M. Jing et al.

    A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

    Nat. Biotechnol.

    (2018)
  • G. Katona et al.

    Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

    Nat. Methods

    (2012)
  • View full text