Multifocal microscopy for functional imaging of neural systems.

Significance: Rapid acquisition of large imaging volumes with microscopic resolution is an essential unmet need in biological research, especially for monitoring rapid dynamical processes such as fast activity in distributed neural systems. Aim: We present a multifocal strategy for fast, volumetric, diffraction-limited resolution imaging over relatively large and scalable fields of view (FOV) using single-camera exposures. Approach: Our multifocal microscopy approach leverages diffraction to image multiple focal depths simultaneously. It is based on a custom-designed diffractive optical element suited to low magnification and large FOV applications and customized prisms for chromatic correction, allowing for wide bandwidth fluorescence imaging. We integrate this system within a conventional microscope and demonstrate that our design can be used flexibly with a variety of magnification/numerical aperture (NA) objectives. Results: We first experimentally and numerically validate this system for large FOV microscope imaging (three orders-of-magnitude larger volumes than previously shown) at resolutions compatible with cellular imaging. We then demonstrate the utility of this approach by visualizing high resolution three-dimensional (3D) distributed neural network at volume rates up to 100 Hz. These demonstrations use genetically encoded Ca 2 + indicators to measure functional neural imaging both in vitro and in vivo. Finally, we explore its potential in other important applications, including blood flow visualization and real-time, microscopic, volumetric rendering. Conclusions: Our study demonstrates the advantage of diffraction-based multifocal imaging techniques for 3D imaging of mm-scale objects from a single-camera exposure, with important applications in functional neural imaging and other areas benefiting from volumetric imaging.

Two-photon imaging of excitatory and inhibitory neural response to infrared neural stimulation.

Significance: Pulsed infrared neural stimulation (INS, 1875 nm) is an emerging neurostimulation technology that delivers focal pulsed heat to activate functionally specific mesoscale networks and holds promise for clinical application. However, little is known about its effect on excitatory and inhibitory cell types in cerebral cortex. Aim: Estimates of summed population neuronal response time courses provide a potential basis for neural and hemodynamic signals described in other studies. Approach: Using two-photon calcium imaging in mouse somatosensory cortex, we have examined the effect of INS pulse train application on hSyn neurons and mDlx neurons tagged with GCaMP6s. Results: We find that, in anesthetized mice, each INS pulse train reliably induces robust response in hSyn neurons exhibiting positive going responses. Surprisingly, mDlx neurons exhibit negative going responses. Quantification using the index of correlation illustrates responses are reproducible, intensity-dependent, and focal. Also, a contralateral activation is observed when INS applied. Conclusions: In sum, the population of neurons stimulated by INS includes both hSyn and mDlx neurons; within a range of stimulation intensities, this leads to overall excitation in the stimulated population, leading to the previously observed activations at distant post-synaptic sites.

Model-based correction of rapid thermal confounds in fluorescence neuroimaging of targeted perturbation.

Significance: An array of techniques for targeted neuromodulation is emerging, with high potential in brain research and therapy. Calcium imaging or other forms of functional fluorescence imaging are central solutions for monitoring cortical neural responses to targeted neuromodulation, but often are confounded by thermal effects that are inter-mixed with neural responses. Aim: Here, we develop and demonstrate a method for effectively suppressing fluorescent thermal transients from calcium responses. Approach: We use high precision phased-array 3 MHz focused ultrasound delivery integrated with fiberscope-based widefield fluorescence to monitor cortex-wide calcium changes. Our approach for detecting the neural activation first takes advantage of the high inter-hemispheric correlation of resting state Ca2+ dynamics and then removes the ultrasound-induced thermal effect by subtracting its simulated spatio-temporal signature from the processed profile. Results: The focused 350 µm-sized ultrasound stimulus triggered rapid localized activation events dominated by transient thermal responses produced by ultrasound. By employing bioheat equation to model the ultrasound heat deposition, we can recover putative neural responses to ultrasound. Conclusions: The developed method for canceling transient thermal fluorescence quenching could also find applications with optical stimulation techniques to monitor thermal effects and disentangle them from neural responses. This approach may help deepen our understanding of the mechanisms and macroscopic effects of ultrasound neuromodulation, further paving the way for tailoring the stimulation regimes toward specific applications.

Shedding light on ultrasound in action: Optical and optoacoustic monitoring of ultrasound brain interventions.

Monitoring brain responses to ultrasonic interventions is becoming an important pillar of a growing number of applications employing acoustic waves to actuate and cure the brain. Optical interrogation of living tissues provides a unique means for retrieving functional and molecular information related to brain activity and disease-specific biomarkers. The hybrid optoacoustic imaging methods have further enabled deep-tissue imaging with optical contrast at high spatial and temporal resolution. The marriage between light and sound thus brings together the highly complementary advantages of both modalities toward high precision interrogation, stimulation, and therapy of the brain with strong impact in the fields of ultrasound neuromodulation, gene and drug delivery, or noninvasive treatments of neurological and neurodegenerative disorders. In this review, we elaborate on current advances in optical and optoacoustic monitoring of ultrasound interventions. We describe the main principles and mechanisms underlying each method before diving into the corresponding biomedical applications. We identify areas of improvement as well as promising approaches with clinical translation potential.

Multimodal Noninvasive Functional Neurophotonic Imaging of Murine Brain-Wide Sensory Responses.

Modern optical neuroimaging approaches are expanding the ability to elucidate complex brain function. Diverse imaging contrasts enable direct observation of neural activity with functional sensors along with the induced hemodynamic responses. To date, decoupling the complex interplay of neurovascular coupling and dynamical physiological states has remained challenging when employing single-modality functional neuroimaging readings. A hybrid fluorescence optoacoustic tomography platform combined with a custom data processing pipeline based on statistical parametric mapping is devised, attaining the first noninvasive observation of simultaneous calcium and hemodynamic activation patterns using optical contrasts. Correlated changes in the oxy- and deoxygenated hemoglobin, total hemoglobin, oxygen saturation, and rapid GCaMP6f fluorescence signals are observed in response to peripheral sensory stimulation. While the concurrent epifluorescence serves to corroborate and complement the functional optoacoustic observations, the latter further aids in decoupling the rapid calcium responses from the slowly varying background in the fluorescence recordings mediated by hemodynamic changes. The hybrid imaging platform expands the capabilities of conventional neuroimaging methods to provide more comprehensive functional readings for studying neurovascular and neurometabolic coupling mechanisms and related diseases.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

High-resolution fluorescence-guided transcranial ultrasound mapping in the live mouse brain.

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance­guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound's localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

Spherical Array System for High-Precision Transcranial Ultrasound Stimulation and Optoacoustic Imaging in Rodents.

Ultrasound can be delivered transcranially to ablate brain tissue, open the blood-brain barrier, or affect neural activity. Transcranial focused ultrasound in small rodents is typically done with low-frequency single-element transducers, which results in unspecific targeting and impedes the concurrent use of fast neuroimaging methods. In this article, we devised a wide-angle spherical array bidirectional interface for high-resolution parallelized optoacoustic imaging and transcranial ultrasound (POTUS) delivery in the same target regions. The system operates between 3 and 9 MHz, allowing to generate and steer focal spots with widths down to [Formula: see text] across a field of view covering the entire mouse brain, while the same array is used to capture high-resolution 3-D optoacoustic data in real time. We showcase the system's versatile beam-forming capacities as well as volumetric optoacoustic imaging capabilities and discuss its potential to noninvasively monitor brain activity and various effects of ultrasound emission.

Publisher Correction: Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Precise Holographic Manipulation of Olfactory Circuits Reveals Coding Features Determining Perceptual Detection.

Sensory systems transform the external world into time-varying spike trains. What features of spiking activity are used to guide behavior? In the mouse olfactory bulb, inhalation of different odors leads to changes in the set of neurons activated, as well as when neurons are activated relative to each other (synchrony) and the onset of inhalation (latency). To explore the relevance of each mode of information transmission, we probed the sensitivity of mice to perturbations across each stimulus dimension (i.e., rate, synchrony, and latency) using holographic two-photon optogenetic stimulation of olfactory bulb neurons with cellular and single-action-potential resolution. We found that mice can detect single action potentials evoked synchronously across <20 olfactory bulb neurons. Further, we discovered that detection depends strongly on the synchrony of activation across neurons, but not the latency relative to inhalation.

Manipulating synthetic optogenetic odors reveals the coding logic of olfactory perception.

How does neural activity generate perception? Finding the combinations of spatial or temporal activity features (such as neuron identity or latency) that are consequential for perception remains challenging. We trained mice to recognize synthetic odors constructed from parametrically defined patterns of optogenetic activation, then measured perceptual changes during extensive and controlled perturbations across spatiotemporal dimensions. We modeled recognition as the matching of patterns to learned templates. The templates that best predicted recognition were sequences of spatially identified units, ordered by latencies relative to each other (with minimal effects of sniff). Within templates, individual units contributed additively, with larger contributions from earlier-activated units. Our synthetic approach reveals the fundamental logic of the olfactory code and provides a general framework for testing links between sensory activity and perception.

Cortical hemodynamic responses induced by low-intensity transcranial ultrasound stimulation of mouse cortex.

Ultrasound-mediated neuromodulation is emerging as a key technology for targeted noninvasive brain stimulation, but key insights into its effects and dose-response characteristics are still missing. The purpose of this study is to systematically evaluate the effect of low-intensity transcranial ultrasound stimulation (TUS) on complementary aspects of cerebral hemodynamic. We simultaneously record the EMG signal, local field potential (LFP) and cortical blood flow (CBF) using electrophysiological recording and laser speckle contrast imaging under ultrasound stimulation to simultaneously monitor motor responses, neural activities and hemodynamic changes during the application of low-intensity TUS in mouse motor cortex, using excitation pulses which caused whisker and tail movement. Our experimental results demonstrate interdependent TUS-induced motor, neural activity and hemodynamic responses that peak approximately 0.55s, 1.05s and 2.5s after TUS onset, respectively, and show a linear coupling relationship between their respective varying response amplitudes to repeated stimuli. We also found monotonic dose-response parametric relations of the CBF peak value increase as a function of stimulation intensity and duration, while stimulus duty-cycle had only a weak effect on peak responses. These findings demonstrate that TUS induces a change in cortical hemodynamics and LSCI provide a high temporal resolution view of these changes.

Isolated Murine Brain Model for Large-Scale Optoacoustic Calcium Imaging.

Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with existing neuroimaging methods having limited capacity when it comes to imaging large tissue volumes at high speeds. Optoacoustic imaging has been shown to be capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep within the mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as a lack of activity labels excitable in the near-infrared window. We have developed and validated an isolated whole mouse brain preparation labeled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions. An optoacoustic imaging system coupled to a superfusion system was further designed and used for rapid volumetric monitoring of stimulus-evoked calcium dynamics in the brain. These new imaging setup and isolated preparation's protocols and characteristics are described here in detail. Our new technique captures calcium fluxes as true three-dimensional information across the entire brain with temporal resolution of 10 ms and spatial resolution of 150 µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with any existing neuroimaging techniques.

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

A genetically encoded near-infrared fluorescent calcium ion indicator.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.

Induced neuro-vascular interactions robustly enhance functional attributes of engineered neural implants.

Engineered neural implants have a myriad of potential basic science and clinical neural repair applications. Although there are implants that are currently undergoing their first clinical investigations, optimizing their long-term viability and efficacy remain an open challenge. Functional implants with pre-vascularization of various engineered tissues have proven to enhance post-implantation host integration, and well-known synergistic neural-vascular interplays suggest that this strategy could also be promising for neural tissue engineering. Here, we report the development of a novel bio-engineered neuro-vascular co-culture construct, and demonstrate that it exhibits enhanced neurotrophic factor expression, and more complex neuronal morphology. Crucially, by introducing genetically encoded calcium indicators (GECIs) into the co-culture, we are able to monitor functional activity of the neural network, and demonstrate greater activity levels and complexity as a result of the introduction of endothelial cells in the construct. The presence of this enhanced activity could putatively lead to superior integration outcomes. Indeed, leveraging on the ability to monitor the construct's development post-implantation with GECIs, we observe improved integration phenotypes in the spinal cord of mice relative to non-vascularized controls. Our approach provides a new experimental system with functional neural feedback for studying the interplay between vascular and neural development while advancing the optimization of neural implants towards potential clinical applications.

Phase-controlled, speckle-free holographic projection with applications in precision optogenetics.

Holographic speckle is a major impediment to computer-generated holographic (CGH) projections in applications ranging from display, optical tweezers, and machining to optogenetic neural control. We present an iterative phase retrieval algorithm that allows the projection of amplitude-controlled speckle-free one-dimensional patterns with a high degree of pattern uniformity. The algorithm, termed the weighted Gerchberg-Saxton with phase-control (GSW-PC), is shown to have the ability to simultaneously control both the phase and amplitude of projected patterns with high diffraction efficiencies. Furthermore, we show that the framework can address the challenge of projecting volumetric phase and amplitude-controlled patterns, by incorporating GSW-PC with the angular spectrum method. The algorithms' performance is numerically and experimentally tested, and further compared with conventional and modern CGH techniques.

Utah optrode array customization using stereotactic brain atlases and 3-D CAD modeling for optogenetic neocortical interrogation in small rodents and nonhuman primates.

As the optogenetic field expands, the need for precise targeting of neocortical circuits only grows more crucial. This work demonstrates a technique for using Solidworks® computer-aided design (CAD) and readily available stereotactic brain atlases to create a three-dimensional (3-D) model of the dorsal region of area visual cortex 4 (V4D) of the macaque monkey (Macaca fascicularis) visual cortex. The 3-D CAD model of the brain was used to customize an [Formula: see text] Utah optrode array (UOA) after it was determined that a high-density ([Formula: see text]) UOA caused extensive damage to marmoset (Callithrix jacchus) primary visual cortex as assessed by electrophysiological recording of spiking activity through a 1.5-mm-diameter through glass via. The [Formula: see text] UOA was customized for optrode length ([Formula: see text]), optrode width ([Formula: see text]), optrode pitch ([Formula: see text]), backplane thickness ([Formula: see text]), and overall form factor ([Formula: see text]). Two [Formula: see text] UOAs were inserted into layer VI of macaque V4D cortices with minimal damage as assessed in fixed tissue cytochrome oxidase staining in nonrecoverable surgeries. Additionally, two [Formula: see text] arrays were implanted in mice (Mus musculus) motor cortices, providing early evidence for long-term tolerability (over 6 months), and for the ability to integrate the UOA with a Holobundle light delivery system toward patterned optogenetic stimulation of cortical networks.

Functional optoacoustic neuro-tomography of calcium fluxes in adult zebrafish brain in vivo.

Genetically-encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping of the activity of entire neuronal populations in vivo. Visualization of these powerful activity sensors has to date been limited to depth-restricted microscopic studies due to intense light scattering in the brain. We demonstrate, for the first time, in vivo real-time volumetric optoacoustic monitoring of calcium transients in adult transgenic zebrafish expressing the GCaMP5G calcium indicator. Fast changes in optoacoustic traces associated with GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques.