High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.

Conduction-Dominated Cryomesh for Organism Vitrification.

Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105  °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.

Strategy dependent recruitment of distributed cortical circuits during spatial navigation.

Spatial navigation is a complex cognitive process that involves neural computations in distributed regions of the brain. Little is known about how cortical regions are coordinated when animals navigate novel spatial environments or how that coordination changes as environments become familiar. We recorded mesoscale calcium (Ca2+) dynamics across large swathes of the dorsal cortex in mice solving the Barnes maze, a 2D spatial navigation task where mice used random, serial, and spatial search strategies to navigate to the goal. Cortical dynamics exhibited patterns of repeated calcium activity with rapid and abrupt shifts between cortical activation patterns at sub-second time scales. We used a clustering algorithm to decompose the spatial patterns of cortical calcium activity in a low dimensional state space, identifying 7 states, each corresponding to a distinct spatial pattern of cortical activation, sufficient to describe the cortical dynamics across all the mice. When mice used serial or spatial search strategies to navigate to the goal, the frontal regions of the cortex were reliably activated for prolonged durations of time (> 1s) shortly after trial initiation. These frontal cortex activation events coincided with mice approaching the edge of the maze from the center and were preceded by temporal sequences of cortical activation patterns that were distinct for serial and spatial search strategies. In serial search trials, frontal cortex activation events were preceded by activation of the posterior regions of the cortex followed by lateral activation of one hemisphere. In spatial search trials, frontal cortical events were preceded by activation of posterior regions of the cortex followed by broad activation of the lateral regions of the cortex. Our results delineated cortical components that differentiate goal- and non-goal oriented spatial navigation strategies.

Distinct mesoscale cortical dynamics encode search strategies during spatial navigation.

Spatial navigation is a complex cognitive process that involves neural computations in distributed regions of the brain. Little is known about how cortical regions are coordinated when animals navigate novel spatial environments or how that coordination changes as environments become familiar. We recorded mesoscale calcium (Ca2+) dynamics across large swathes of the dorsal cortex in mice solving the Barnes maze, a 2D spatial navigation task where mice used random, serial, and spatial search strategies to navigate to the goal. Cortical dynamics exhibited patterns of repeated calcium activity with rapid and abrupt shifts between cortical activation patterns at sub-second time scales. We used a clustering algorithm to decompose the spatial patterns of cortical calcium activity in a low dimensional state space, identifying 7 states, each corresponding to a distinct spatial pattern of cortical activation, sufficient to describe the cortical dynamics across all the mice. When mice used serial or spatial search strategies to navigate to the goal, the frontal regions of the cortex were reliably activated for prolonged durations of time (> 1s) shortly after trial initiation. These frontal cortex activation events coincided with mice approaching the edge of the maze from the center and were preceded by temporal sequences of cortical activation patterns that were distinct for serial and spatial search strategies. In serial search trials, frontal cortex activation events were preceded by activation of the posterior regions of the cortex followed by lateral activation of one hemisphere. In spatial search trials, frontal cortical events were preceded by activation of posterior regions of the cortex followed by broad activation of the lateral regions of the cortex. Our results delineated cortical components that differentiate goal- and non-goal oriented spatial navigation strategies.

Fully desktop fabricated flexible graphene electrocorticography (ECoG) arrays.

Objective:Flexible Electrocorticography (ECoG) electrode arrays that conform to the cortical surface and record surface field potentials from multiple brain regions provide unique insights into how computations occurring in distributed brain regions mediate behavior. Specialized microfabrication methods are required to produce flexible ECoG devices with high-density electrode arrays. However, these fabrication methods are challenging for scientists without access to cleanroom fabrication equipment.Results:Here we present a fully desktop fabricated flexible graphene ECoG array. First, we synthesized a stable, conductive ink via liquid exfoliation of Graphene in Cyrene. Next, we established a stencil-printing process for patterning the graphene ink via laser-cut stencils on flexible polyimide substrates. Benchtop tests indicate that the graphene electrodes have good conductivity of ∼1.1 × 103S cm-1, flexibility to maintain their electrical connection under static bending, and electrochemical stability in a 15 d accelerated corrosion test. Chronically implanted graphene ECoG devices remain fully functional for up to 180 d, with averagein vivoimpedances of 24.72 ± 95.23 kΩ at 1 kHz. The ECoG device can measure spontaneous surface field potentials from mice under awake and anesthetized states and sensory stimulus-evoked responses.Significance:The stencil-printing fabrication process can be used to create Graphene ECoG devices with customized electrode layouts within 24 h using commonly available laboratory equipment.

Wide-field calcium imaging reveals widespread changes in cortical functional connectivity following mild traumatic brain injury in the mouse.

>2.5 million individuals in the United States suffer mild traumatic brain injuries (mTBI) annually. Mild TBI is characterized by a brief period of altered consciousness, without objective findings of anatomic injury on clinical imaging or physical deficit on examination. Nevertheless, a subset of mTBI patients experience persistent subjective symptoms and repeated mTBI can lead to quantifiable neurological deficits, suggesting that each mTBI alters neurophysiology in a deleterious manner not detected using current clinical methods. To better understand these effects, we performed mesoscopic Ca2+ imaging in mice to evaluate how mTBI alters patterns of neuronal interactions across the dorsal cerebral cortex. Spatial Independent Component Analysis (sICA) and Localized semi-Nonnegative Matrix Factorization (LocaNMF) were used to quantify changes in cerebral functional connectivity (FC). Repetitive, mild, controlled cortical impacts induce temporary neuroinflammatory responses, characterized by increased density of microglia exhibiting de-ramified morphology. These temporary neuro-inflammatory changes were not associated with compromised cognitive performance in the Barnes maze or motor function as assessed by rotarod. However, long-term alterations in functional connectivity (FC) were observed. Widespread, bilateral changes in FC occurred immediately following impact and persisted for up to 7 weeks, the duration of the experiment. Network alterations include decreases in global efficiency, clustering coefficient, and nodal strength, thereby disrupting functional interactions and information flow throughout the dorsal cerebral cortex. A subnetwork analysis shows the largest disruptions in FC were concentrated near the impact site. Therefore, mTBI induces a transient neuroinflammation, without alterations in cognitive or motor behavior, and a reorganized cortical network evidenced by the widespread, chronic alterations in cortical FC.

Wide-Field Calcium Imaging of Neuronal Network Dynamics In Vivo.

A central tenet of neuroscience is that sensory, motor, and cognitive behaviors are generated by the communications and interactions among neurons, distributed within and across anatomically and functionally distinct brain regions. Therefore, to decipher how the brain plans, learns, and executes behaviors requires characterizing neuronal activity at multiple spatial and temporal scales. This includes simultaneously recording neuronal dynamics at the mesoscale level to understand the interactions among brain regions during different behavioral and brain states. Wide-field Ca2+ imaging, which uses single photon excitation and improved genetically encoded Ca2+ indicators, allows for simultaneous recordings of large brain areas and is proving to be a powerful tool to study neuronal activity at the mesoscopic scale in behaving animals. This review details the techniques used for wide-field Ca2+ imaging and the various approaches employed for the analyses of the rich neuronal-behavioral data sets obtained. Also discussed is how wide-field Ca2+ imaging is providing novel insights into both normal and altered neural processing in disease. Finally, we examine the limitations of the approach and new developments in wide-field Ca2+ imaging that are bringing new capabilities to this important technique for investigating large-scale neuronal dynamics.

Polymer Skulls With Integrated Transparent Electrode Arrays for Cortex-Wide Opto-Electrophysiological Recordings.

Electrophysiology and optical imaging provide complementary neural sensing capabilities - electrophysiological recordings have high temporal resolution, while optical imaging allows recording of genetically-defined populations at high spatial resolution. Combining these two modalities for simultaneous large-scale, multimodal sensing of neural activity across multiple brain regions can be very powerful. Here, transparent, inkjet-printed electrode arrays with outstanding optical and electrical properties are seamlessly integrated with morphologically conformant transparent polymer skulls. Implanted on transgenic mice expressing the Calcium (Ca2+ ) indicator GCaMP6f in excitatory neurons, these "eSee-Shells" provide a robust opto-electrophysiological interface for over 100 days. eSee-Shells enable simultaneous mesoscale Ca2+ imaging and electrocorticography (ECoG) acquisition from multiple brain regions covering 45 mm2 of cortex under anesthesia and in awake animals. The clarity and transparency of eSee-Shells allow recording single-cell Ca2+ signals directly below the electrodes and interconnects. Simultaneous multimodal measurement of cortical dynamics reveals changes in both ECoG and Ca2+ signals that depend on the behavioral state.

Wide-Field Calcium Imaging of Dynamic Cortical Networks during Locomotion.

Motor behavior results in complex exchanges of motor and sensory information across cortical regions. Therefore, fully understanding the cerebral cortex's role in motor behavior requires a mesoscopic-level description of the cortical regions engaged, their functional interactions, and how these functional interactions change with behavioral state. Mesoscopic Ca2+ imaging through transparent polymer skulls in mice reveals elevated activation of the dorsal cerebral cortex during locomotion. Using the correlations between the time series of Ca2+ fluorescence from 28 regions (nodes) obtained using spatial independent component analysis (sICA), we examined the changes in functional connectivity of the cortex from rest to locomotion with a goal of understanding the changes to the cortical functional state that facilitate locomotion. Both the transitions from rest to locomotion and from locomotion to rest show marked increases in correlation among most nodes. However, once a steady state of continued locomotion is reached, many nodes, including primary motor and somatosensory nodes, show decreases in correlations, while retrosplenial and the most anterior nodes of the secondary motor cortex show increases. These results highlight the changes in functional connectivity in the cerebral cortex, representing a series of changes in the cortical state from rest to locomotion and on return to rest.

Multiscale, multi-perspective imaging assisted robotic microinjection of 3D biological structures.

Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.

Miniaturized head-mounted microscope for whole-cortex mesoscale imaging in freely behaving mice.

The advent of genetically encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive-index-matched skulls, has enabled mesoscale cortical activity imaging in head-fixed mice. However, neural activity during unrestrained behavior substantially differs from neural activity in head-fixed animals. For whole-cortex imaging in freely behaving mice, we present the 'mini-mScope', a widefield, miniaturized, head-mounted fluorescence microscope that is compatible with transparent polymer skull preparations. With a field of view of 8 × 10 mm2 and weighing less than 4 g, the mini-mScope can image most of the mouse dorsal cortex with resolutions ranging from 39 to 56 µm. We used the mini-mScope to record mesoscale calcium activity across the dorsal cortex during sensory-evoked stimuli, open field behaviors, social interactions and transitions from wakefulness to sleep.

Through the looking glass: A review of cranial window technology for optical access to the brain.

Deciphering neurologic function is a daunting task, requiring understanding the neuronal networks and emergent properties that arise from the interactions among single neurons. Mechanistic insights into neuronal networks require tools that simultaneously assess both single neuron activity and the consequent mesoscale output. The development of cranial window technologies, in which the skull is thinned or replaced with a synthetic optical interface, has enabled monitoring neuronal activity from subcellular to mesoscale resolution in awake, behaving animals when coupled with advanced microscopy techniques. Here we review recent achievements in cranial window technologies, appraise the relative merits of each design and discuss the future research in cranial window design.

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

Assembly and operation of an open-source, computer numerical controlled (CNC) robot for performing cranial microsurgical procedures.

Cranial microsurgery is an essential procedure for accessing the brain through the skull that can be used to introduce neural probes that measure and manipulate neural activity. Neuroscientists have typically used tools such as high-speed drills adapted from dentistry to perform these procedures. As the number of technologies available for neuroscientists has increased, the corresponding cranial microsurgery procedures to deploy them have become more complex. Using a robotic tool that automatically performs these procedures could standardize cranial microsurgeries across neuroscience laboratories and democratize the more challenging procedures. We have recently engineered a robotic surgery platform that utilizes principles of computer numerical control (CNC) machining to perform a wide variety of automated cranial procedures. Here, we describe how to adapt, configure and use an inexpensive desktop CNC mill equipped with a custom-built surface profiler for performing CNC-guided microsurgery on mice. Detailed instructions are provided to utilize this 'Craniobot' for performing circular craniotomies for coverslip implantation, large craniotomies for implanting transparent polymer skulls for cortex-wide imaging access and skull thinning for intact skull imaging. The Craniobot can be set up in <2 weeks using parts that cost <$1,500, and we anticipate that the Craniobot could be easily adapted for use in other small animals.

Robotic platform for microinjection into single cells in brain tissue.

Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low-throughput process. We developed the "Autoinjector", a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high-performance single-cell manipulation technique and will provide a powerful new platform for performing single-cell analyses in tissue for bioengineering and biophysics applications.

Autonomous patch-clamp robot for functional characterization of neurons in vivo: development and application to mouse visual cortex.

Patch clamping is the gold standard measurement technique for cell-type characterization in vivo, but it has low throughput, is difficult to scale, and requires highly skilled operation. We developed an autonomous robot that can acquire multiple consecutive patch-clamp recordings in vivo. In practice, 40 pipettes loaded into a carousel are sequentially filled and inserted into the brain, localized to a cell, used for patch clamping, and disposed. Automated visual stimulation and electrophysiology software enables functional cell-type classification of whole cell-patched cells, as we show for 37 cells in the anesthetized mouse in visual cortex (V1) layer 5. We achieved 9% yield, with 5.3 min per attempt over hundreds of trials. The highly variable and low-yield nature of in vivo patch-clamp recordings will benefit from such a standardized, automated, quantitative approach, allowing development of optimal algorithms and enabling scaling required for large-scale studies and integration with complementary techniques. NEW & NOTEWORTHY In vivo patch-clamp is the gold standard for intracellular recordings, but it is a very manual and highly skilled technique. The robot in this work demonstrates the most automated in vivo patch-clamp experiment to date, by enabling production of multiple, serial intracellular recordings without human intervention. The robot automates pipette filling, wire threading, pipette positioning, neuron hunting, break-in, delivering sensory stimulus, and recording quality control, enabling in vivo cell-type characterization.

Cortex-wide neural interfacing via transparent polymer skulls.

Neural computations occurring simultaneously in multiple cerebral cortical regions are critical for mediating behaviors. Progress has been made in understanding how neural activity in specific cortical regions contributes to behavior. However, there is a lack of tools that allow simultaneous monitoring and perturbing neural activity from multiple cortical regions. We engineered 'See-Shells'-digitally designed, morphologically realistic, transparent polymer skulls that allow long-term (>300 days) optical access to 45 mm2 of the dorsal cerebral cortex in the mouse. We demonstrate the ability to perform mesoscopic imaging, as well as cellular and subcellular resolution two-photon imaging of neural structures up to 600 µm deep. See-Shells allow calcium imaging from multiple, non-contiguous regions across the cortex. Perforated See-Shells enable introducing penetrating neural probes to perturb or record neural activity simultaneously with whole cortex imaging. See-Shells are constructed using common desktop fabrication tools, providing a powerful tool for investigating brain structure and function.

Craniobot: A computer numerical controlled robot for cranial microsurgeries.

Over the last few decades, a plethora of tools has been developed for neuroscientists to interface with the brain. Implementing these tools requires precisely removing sections of the skull to access the brain. These delicate cranial microsurgical procedures need to be performed on the sub-millimeter thick bone without damaging the underlying tissue and therefore, require significant training. Automating some of these procedures would not only enable more precise microsurgical operations, but also facilitate widespread use of advanced neurotechnologies. Here, we introduce the "Craniobot", a cranial microsurgery platform that combines automated skull surface profiling with a computer numerical controlled (CNC) milling machine to perform a variety of cranial microsurgical procedures on mice. The Craniobot utilizes a low-force contact sensor to profile the skull surface and uses this information to perform precise milling operations within minutes. We have used the Craniobot to perform intact skull thinning and open small to large craniotomies over the dorsal cortex.

Automated Assessment of Loss of Consciousness Using Whisker And Paw Movements During Anesthetic Dosing in Head-Fixed Rodents.

The precise identification of loss of consciousness (LOC) is key to studying the effects of anesthetic drugs in neural systems. The standard behavioral assay for identifying LOC in rodents is the Loss of Righting Reflex (LORR), assessed by placing the animal in the supine position every minute until it fails to right itself. However, this assay cannot be used when the rodents are head-fixed, which limits the use of powerful techniques such as multi-electrode recordings, in vivo patch clamp, and neuronal imaging. In these situations, an alternative way to assess LOC is needed. We propose that loss of movement (LOM) in whiskers and paws of head-fixed animals can be used as an alternative behavioral assay in head-fixed animals. Unlike LORR, LOM in whiskers and paws is much harder to detect by visual inspection. Therefore, we developed a method to automatically assess for LOM of whiskers and paws in head fixed rodents during in vivo patch clamp recordings. Our method uses an algorithm based on optical flow and point-process filtering which can be run on images acquired on regular cameras at low frame-rates. We show that the algorithm can achieve at least comparable accuracy in detecting LOC when compared with consensus among human observers, as well as improved precision when compared with individual observers. In the future, we aim to to expand the method to detect more behavioral end-points during anesthesia such as paradoxical excitation. Eventually, we hope to enable multi-modal anesthesia studies, which incorporates behavioral and neurophysiological data.