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Cellular Resolution Imaging of Neuronal Activity Across Space and Time in the Mammalian Brain Mitchell Clough1 and Jerry L

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Cellular Resolution Imaging of Neuronal Activity Across Space and Time in the Mammalian Brain Mitchell Clough1 and Jerry L Available online at www.sciencedirect.com Current Opinion in ScienceDirect Biomedical Engineering Cellular resolution imaging of neuronal activity across space and time in the mammalian brain Mitchell Clough1 and Jerry L. Chen1,2,3 Abstract Compared with electrophysiological methods, optical The action potential has long been understood to be the methods to noninvasively measure population activity fundamental bit of information in brain, but how these spikes provide distinct advantages in the ability to obtain encode representations of stimuli and drive behavior remains precise spatial information, enabling the multimodal unclear. Large-scale neuronal recordings with cellular and mapping of gene expression [1] and connectivity [2] spike-time resolution spanning multiple brain regions are that is critical for the dissection of circuit function. needed to capture relevant network dynamics that can be Continuing advancements in genetically encoded fluo- sparse and distributed across the population. This review fo- rescent activity sensors have driven the development of cuses on recent advancements in optical methods that have both one-photon and multiphoton microscopy for pushed the boundaries for simultaneous population recordings simultaneous population recordings at increasing vol- at increasing volumes, distances, depths, and speeds. The umes, distances, depths, and speeds. This review will integration of these technologies will be critical for overcoming focus on recent advancements in microscopy techniques fundamental limits in the pursuit of whole brain imaging in that have pushed the boundaries across these different mammalian species. dimensions and discuss the prospects for potentially imaging activity across the entire mammalian brain. Addresses 1 Department of Biomedical Engineering, Boston University, Boston, Whole mammalian brain imaging: USA 2 specifications and challenges Department of Biology, Boston University, Boston, USA What would it take to image the activity of every single 3 Center for Neurophotonics, Boston University, Boston, USA neuron in a mammalian brain? The engineering chal- Corresponding author: Chen, Jerry L ([email protected]) lenges are best understood by considering the physical requirements and the biological constraints. Develop- ment of optical systems has been focused on imaging Current Opinion in Biomedical Engineering 2019, 12:95–101 the mouse brain which is composed of w75 million This review comes from a themed issue on Neural Engineering: High neurons that occupy a volume of w500 mm3. To resolve Resolution Cell Imaging the cell body of each neuron, an optical resolution of Edited by John A White and Xue Han 5 mm is needed. One fundamental challenge is developing optical systems that are capable of imaging https://doi.org/10.1016/j.cobme.2019.11.004 large volumes of tissue at high spatial resolution. The second fundamental challenge is the imaging speed 2468-4511/© 2019 Elsevier Inc. All rights reserved. required to capture population dynamics. The necessary speed depends on the signals being measured and the indicators of activity being used. The membrane action Introduction potential of a neuron is the direct measure of a neuron’s To understand how the brain is capable of carrying activity. Detecting an action potential requires w1ms out a certain behavior, it is necessary to determine temporal resolution. Combining the temporal and the underlying neural code that can generate com- spatial resolutions required along with the volume of the 3 plex cognitive thought. To characterize the network brain means imaging 500,000 mm /s at 5 mm resolution activity patterns that form the basis of such com- to capture all the action potentials of the mouse brain. putations, the ability to measure information More modest speeds are sufficient when using calcium processing at its native speed and fidelity in a signals as a proxy for action potential firing activity. comprehensive manner across all participating neu- Calcium influx into the neuron occurs on the order of rons has become both a scientific and engineering w100 ms, which translates to volume imaging rates of 3 goal in neuroscience. Technologies for large-scale 5,000 mm /s. simultaneous recordings of neuronal populations spanning an entire brain will inform us how our Beyond the spatial and temporal requirements, a major nervous system represents the external environment, consideration is the photon budget involved in exciting formsandrecallsmemories,makesdecisions,and the fluorescent calcium or voltage indicators. Detection carries out actions. of action potentialerelated events is determined by the overall brightness, activity-related change in www.sciencedirect.com Current Opinion in Biomedical Engineering 2019, 12:95–101 96 Neural Engineering: High Resolution Cell Imaging fluorescence, kinetics, and availability of the indicator applications for comprehensively measuring local circuit [3e5]. Current calcium indicators are expressed in the dynamics and understanding functional relationships in cytosol, filling the volume of the neuron, with transients the architectural organization of brain areas. Several on the order of 100 milliseconds and signal changes up to methods using temporally or spatially structured illu- 10-fold above baseline. Depending on how well the mination have been applied to increase imaging volume timing of spike-related events needs to be resolved, while maintaining sufficient temporal resolution. relevant calcium signals can be recorded at a rate in the Widefield one-photon fluorescence microscopy has range of w1e30 Hz and at an average power of less than traditionally provided large field of view imaging without 30 mW per voxel. In contrast, voltage indicators need to scanning. Because of the lack of spatial confinement, be localized to the cell membrane and possess milli- axial information about the sample is normally not pre- second response times [6,7]. The differences in locali- sent. Toaddress this, modulated-illumination extended- zation and kinetics severely limit the number of depth-of-field imaging (MI-EDOF) can be used to gain available excitable molecules during voltage imaging, axial resolution [10]. This technique makes use of a requiring greater photo-energy to achieve comparable deformable mirror (DM) to increase the depth of field of signal-to-noise as during calcium imaging. However, the a standard widefield imaging setup [11]. The DM allows light energy needed for excitation is ultimately limited for rapidly sweeping the focus of the microscope axially by the amount of light the brain can safely accept which allows in-focus imaging of a whole volume at without producing thermal damage and phototoxicity. frame rates limited by the camera. By controlling the Up to 400 mW at a 33% duty cycle across 1 h can be intensity of the illumination light as a function of the delivered before tissue damage is observed [8]. This focal depth and then using the optical transfer function indicates that an efficient photon budget is needed to to perform deconvolution, the axial position of each achieve high-speed, large-scale excitation. The final pixel in the image can be estimated. major factor is the high degree of light scattering that occurs in mammalian brain tissue which limits the depth Light-sheet microscopy makes use of laser light focused of imaging possible by reducing excitation efficiency as along one axis and detection optics arranged perpen- well as the ability to spatially resolve emitted fluores- dicularly to the excitation light to optically section a cence during detection [9]. volume at high speeds. This is typically accomplished through the use of two separate objectives, one for Imaging neuronal activity of increasing fluorescence excitation and one for fluorescence volumes detection. Although this configuration is amenable to Developing technologies to match the physical and imaging the brains of small organisms such as zebrafish, biological requirements for whole brain imaging is a it presents steric challenges for imaging larger formidable challenge. Individual efforts, thus far, have mammalian brains. One method to address this issue, been focused on pushing the capabilities along a subset called swept confocally-aligned planar excitation of dimensions, which can still be used for specific ex- (SCAPE) [12,13], makes use of oblique plane micro- periments (Figure 1). Volumetric imaging has scopy (OPM) [14]. SCAPE allows for 3D volumetric Figure 1 (a) Survey of current imaging systems (numbered references) developed for cellular resolution brain imaging plotted along imaging volume and temporal resolution. (b) Total field of views of current imaging systems. Current Opinion in Biomedical Engineering 2019, 12:95–101 www.sciencedirect.com Imaging neuronal activity Clough and Chen 97 imaging at volume rates of 10 Hz in mice. A downside to the two arms of the PSFand (2) the depth of the neuron the original implementation of SCAPE [12] was that the in the tissue. As a result, the 3D volume is compressed excitation light sheet tilted as a volumetric scan was to a 2D image, but information about the axial position performed. This creates a point-spread-function (PSF) of each neuron is preserved. which is variable across a scan and adds computational complexity to the exact 3D reconstruction of the scan- Imaging neuronal activity at increasing ned volume. More recently, SCAPE 2.0 [13] and another speeds implementation of OPM called scanned oblique plane The development of genetically encoded voltage in- illumination
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