Rapid detection of neurons in widefield calcium imaging datasets after training with synthetic data.

Widefield microscopy can provide optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, tissue scattering and background contamination results in signal deterioration, making the extraction of neuronal activity challenging, laborious and time consuming. Here we present our deep-learning-based widefield neuron finder (DeepWonder), which is trained by simulated functional recordings and effectively works on experimental data to achieve high-fidelity neuronal extraction. Equipped with systematic background contribution priors, DeepWonder conducts neuronal inference with an order-of-magnitude-faster speed and improved accuracy compared with alternative approaches. DeepWonder removes background contaminations and is computationally efficient. Specifically, DeepWonder accomplishes 50-fold signal-to-background ratio enhancement when processing terabytes-scale cortex-wide functional recordings, with over 14,000 neurons extracted in 17 h.

Implanted multichannel microelectrode array for simultaneous electrophysiological signal detection of hippocampal CA1 and DG neurons of simulated microgravity rats.

Microgravity can cause body fluids to accumulate in the brain, resulting in brain damage. There are few studies that focus on the detection of electrophysiological signals in simulated microgravity rats, and the precise mechanisms are unknown. In this study, a new device was established to investigate the influence of microgravity on hippocampal neurons. A 16-channel microelectrode array was fabricated for in vivo multichannel electrophysiological recordings. In these experiments, microelectrode array was inserted into normal, 28-day tail suspension model, and 3-day recovered after modulation rats to record electrophysiological signals in the CA1 and DG regions of the hippocampus. Through analysis of electrophysiological signals, we obtained the following results: (1) spike signals of model rats sporadically showed brief periods of suspension involving most of the recorded neurons, which corresponded to slow and smooth peaks in local field potentials. For model rats, the firing rate was reduced, and the power in the frequency spectrum was concentrated in the slow frequency band (0-1 Hz); (2) after the detected hippocampal cells divided into pyramidal cells and interneurons, the spike duration of pyramidal cells showed remarkable latency, and their average firing rates showed a more significant decrease compared to interneurons. These results demonstrate that the hippocampal neurons were impaired after modulation in the cellular dimension, and pyramidal cells were more susceptible than interneurons.

Bio-electrochemical microelectrode arrays for glutamate and electrophysiology detection in hippocampus of temporal lobe epileptic rats.

Temporal Lobe Epilepsy (TLE) is a chronic neurological disorder, characterized by sudden, repeated and transient central nervous system dysfunction. For better understanding of TLE, bio-nanomodified microelectrode arrays (MEA) are designed, for the achievement of high-quality simultaneous detection of glutamate signals (Glu) and multi-channel electrophysiological signals including action potentials (spikes) and local field potentials (LFPs). The MEA was fabricated by Micro-Electro-Mechanical System fabrication technology and all recording sites were modified with platinum black nano-particles, the average impedance decreased by nearly 90 times. Additionally, glutamate oxidase was also modified for the detection of Glu. The average sensitivity of the electrode in Glu solution was 1.999 ±â€¯0.032 × 10-2pA/µM·µm2(n = 3) and linearity was R = 0.9986, with a good selectivity of 97.82% for glutamate and effective blocking of other interferents. In the in-vivo experiments, the MEA was subjected in hippocampus to electrophysiology and Glu concentration detection. During seizures, the fire rate of spikes increases, and the interspike interval is concentrated within 30 ms. The amplitude of LFPs increases by 3 times and the power increases. The Glu level (4.22 µM, n = 4) was obviously higher than normal rats (2.24 µM, n = 4). The MEA probe provides an advanced tool for the detection of dual-mode signals in the research of neurological diseases.

In Situ Real-Time Monitoring of Glutamate and Electrophysiology from Cortex to Hippocampus in Mice Based on a Microelectrode Array.

Changes in the structure and function of the hippocampus contribute to epilepsy, schizophrenia and other neurological or mental disorders of the brain. Since the function of the hippocampus depends heavily on the glutamate (Glu) signaling pathways, in situ real-time detection of Glu neurotransmitter release and electrophysiological signals in hippocampus is of great significance. To achieve the dual-mode detection in mouse hippocampus in vivo, a 16-channel implantable microelectrode array (MEA) was fabricated by micro-electromechanical system (MEMS) technology. Twelve microelectrode sites were modified with platinum black for electrophysiological recording and four sites were modified with glutamate oxidase (GluOx) and 1,3-phenylenediamine (mPD) for selective electrochemical detection of Glu. The MEA was implanted from cortex to hippocampus in mouse brain for in situ real-time monitoring of Glu and electrophysiological signals. It was found that the Glu concentration in hippocampus was roughly 50 µM higher than that in the cortex, and the firing rate of concurrently recorded spikes declined from 6.32 ± 4.35 spikes/s in cortex to 0.09 ± 0.06 spikes/s in hippocampus. The present results demonstrated that the dual-mode MEA probe was capable in neurological detections in vivo with high spatial resolution and dynamical response, which lays the foundation for further pathology studies in the hippocampus of mouse models with nervous or mental disorders.

Plasmid-encoding vasostatin inhibited the growth and metastasis of human hepatocellular carcinoma cells.

The growth and metastasis of solid tumors depends on angiogenesis. Anti-angiogenesis therapy may represent a promising therapeutic option. Vasostatin, the N-terminal domain of calreticulin, is a very potent endogenous inhibitor of angiogenesis and tumor growth. In this study, we attempted to investigate whether plasmid-encoding vasostatin complexed with cationic liposome could suppress the growth and metastasis of hepatocellular carcinoma in vivo and discover its possible mechanism of action. Apoptosis induction of pSecTag2B-vasostatin plasmid on murine endothelial cells (MS1) was examined by flow cytometric analysis in vitro. Nude mice bearing HCCLM3 tumor received pSecTag2B-vasostatin, pSecTag2B-Null, and 0.9 % NaCl solution, respectively. Tumor net weight was measured and survival time was observed. Microvessel density within tumor tissues was determined by CD31 immunohistochemistry. H&E staining of lungs and TUNEL assay of primary tumor tissues were also conducted. The results displayed that pSecTag2B-vasostatin could inhibit the growth and metastasis of hepatocellular carcinoma xenografts and prolong survival time compared with the controls in vivo. Moreover, histologic analysis revealed that pSecTag2B-vasostatin treatment increased apoptosis and inhibited angiogenesis. The present data may be of importance to the further exploration of this new anti-angiogenesis approach in the treatment of hepatocellular cancer.

Long-term intravital subcellular imaging with confocal scanning light-field microscopy.

Long-term observation of subcellular dynamics in living organisms is limited by background fluorescence originating from tissue scattering or dense labeling. Existing confocal approaches face an inevitable tradeoff among parallelization, resolution and phototoxicity. Here we present confocal scanning light-field microscopy (csLFM), which integrates axially elongated line-confocal illumination with the rolling shutter in scanning light-field microscopy (sLFM). csLFM enables high-fidelity, high-speed, three-dimensional (3D) imaging at near-diffraction-limit resolution with both optical sectioning and low phototoxicity. By simultaneous 3D excitation and detection, the excitation intensity can be reduced below 1 mW mm-2, with 15-fold higher signal-to-background ratio over sLFM. We imaged subcellular dynamics over 25,000 timeframes in optically challenging environments in different species, such as migrasome delivery in mouse spleen, retractosome generation in mouse liver and 3D voltage imaging in Drosophila. Moreover, csLFM facilitates high-fidelity, large-scale neural recording with reduced crosstalk, leading to high orientation selectivity to visual stimuli, similar to two-photon microscopy, which aids understanding of neural coding mechanisms.

Distinct neural activities of the cortical layer 2/3 across isoflurane anesthesia: A large-scale simultaneous observation of neurons.

Anesthesia inhibits neural activity in the brain, causing patients to lose consciousness and sensation during the surgery. Layers 2/3 of the cortex are important structures for the integration of information and consciousness, which are closely related to normal cognitive function. However, the dynamics of the large-scale population of neurons across multiple regions in layer 2/3 during anesthesia and recovery processes remains unclear. We conducted simultaneous observations and analysis of large-scale calcium signaling dynamics across multiple cortical regions within cortical layer 2/3 during isoflurane anesthesia and recovery in vivo by high-resolution wide-field microscopy. Under isoflurane-induced anesthesia, there is an overall decrease in neuronal activity across multiple regions in the cortical layer 2/3. Notably, some neurons display a paradoxical increase in activity during anesthesia. Additionally, the activity among multiple cortical regions under anesthesia was homogeneous. It is only during the recovery phase that variability emerges in the extent of increased neural activity across different cortical regions. Within the same duration of anesthesia, neural activity did not return to preanesthetic levels. To sum up, anesthesia as a dynamic alteration of brain functional networks, encompassing shifts in patterns of neural activity, homogeneousness among cortical neurons and regions, and changes in functional connectivity. Recovery from anesthesia does not entail a reversal of these effects within the same timeframe.

A miniaturized mesoscope for the large-scale single-neuron-resolved imaging of neuronal activity in freely behaving mice.

Exploring the relationship between neuronal dynamics and ethologically relevant behaviour involves recording neuronal-population activity using technologies that are compatible with unrestricted animal behaviour. However, head-mounted microscopes that accommodate weight limits to allow for free animal behaviour typically compromise field of view, resolution or depth range, and are susceptible to movement-induced artefacts. Here we report a miniaturized head-mounted fluorescent mesoscope that we systematically optimized for calcium imaging at single-neuron resolution, for increased fields of view and depth of field, and for robustness against motion-generated artefacts. Weighing less than 2.5 g, the mesoscope enabled recordings of neuronal-population activity at up to 16 Hz, with 4 µm resolution over 300 µm depth-of-field across a field of view of 3.6 × 3.6 mm2 in the cortex of freely moving mice. We used the mesoscope to record large-scale neuronal-population activity in socially interacting mice during free exploration and during fear-conditioning experiments, and to investigate neurovascular coupling across multiple cortical regions.

Neuron populations across layer 2-6 in the mouse visual cortex exhibit different coding abilities in the awake mice.

Introduction: The visual cortex is a key region in the mouse brain, responsible for processing visual information. Comprised of six distinct layers, each with unique neuronal types and connections, the visual cortex exhibits diverse decoding properties across its layers. This study aimed to investigate the relationship between visual stimulus decoding properties and the cortical layers of the visual cortex while considering how this relationship varies across different decoders and brain regions. Methods: This study reached the above conclusions by analyzing two publicly available datasets obtained through two-photon microscopy of visual cortex neuronal responses. Various types of decoders were tested for visual cortex decoding. Results: Our findings indicate that the decoding accuracy of neuronal populations with consistent sizes varies among visual cortical layers for visual stimuli such as drift gratings and natural images. In particular, layer 4 neurons in VISp exhibited significantly higher decoding accuracy for visual stimulus identity compared to other layers. However, in VISm, the decoding accuracy of neuronal populations with the same size in layer 2/3 was higher than that in layer 4, despite the overall accuracy being lower than that in VISp and VISl. Furthermore, SVM surpassed other decoders in terms of accuracy, with the variation in decoding performance across layers being consistent among decoders. Additionally, we found that the difference in decoding accuracy across different imaging depths was not associated with the mean orientation selectivity index (OSI) and the mean direction selectivity index (DSI) neurons, but showed a significant positive correlation with the mean reliability and mean signal-to-noise ratio (SNR) of each layer's neuron population. Discussion: These findings lend new insights into the decoding properties of the visual cortex, highlighting the role of different cortical layers and decoders in determining decoding accuracy. The correlations identified between decoding accuracy and factors such as reliability and SNR pave the way for more nuanced understandings of visual cortex functioning.

Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution.

Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces.

Cerebral cortex and hippocampus neural interaction during vagus nerve stimulation under in vivo large-scale imaging.

Objective: The purpose of this study was to study mechanisms of VNS modulation from a single neuron perspective utilizing a practical observation platform with single neuron resolution and widefield, real-time imaging coupled with an animal model simultaneously exposing the cerebral cortex and the hippocampus. Methods: We utilized the observation platform characterized of widefield of view, real-time imaging, and high spatiotemporal resolution to obtain the neuronal activities in the cerebral cortex and the hippocampus during VNS in awake states and under anesthesia. Results: Some neurons in the hippocampus were tightly related to VNS modulation, and varied types of neurons showed distinct responses to VNS modulation. Conclusion: We utilized such an observation platform coupled with a novel animal model to obtain more information on neuron activities in the cerebral cortex and the hippocampus, providing an effective method to further study the mechanisms of therapeutic effects modulated by VNS.

Real-time denoising enables high-sensitivity fluorescence time-lapse imaging beyond the shot-noise limit.

A fundamental challenge in fluorescence microscopy is the photon shot noise arising from the inevitable stochasticity of photon detection. Noise increases measurement uncertainty and limits imaging resolution, speed and sensitivity. To achieve high-sensitivity fluorescence imaging beyond the shot-noise limit, we present DeepCAD-RT, a self-supervised deep learning method for real-time noise suppression. Based on our previous framework DeepCAD, we reduced the number of network parameters by 94%, memory consumption by 27-fold and processing time by a factor of 20, allowing real-time processing on a two-photon microscope. A high imaging signal-to-noise ratio can be acquired with tenfold fewer photons than in standard imaging approaches. We demonstrate the utility of DeepCAD-RT in a series of photon-limited experiments, including in vivo calcium imaging of mice, zebrafish larva and fruit flies, recording of three-dimensional (3D) migration of neutrophils after acute brain injury and imaging of 3D dynamics of cortical ATP release. DeepCAD-RT will facilitate the morphological and functional interrogation of biological dynamics with a minimal photon budget.

Controlled evaporative self-assembly of poly(3-hexylthiophene) monitored with confocal polarized Raman spectroscopy.

Highly-ordered, parallel gradient P3HT stripes are fabricated through a facile deposition method based on controlled evaporative self-assembly (CESA). Confocal polarized Raman spectroscopy is employed to determine the orientation of P3HT chains in individual stripes and spacing. This is the first report on orientation at the molecular level, beyond sole morphology, in the patterns assembled by CESA. P3HT chains tend to aggregate into one-dimensional nanowhiskers in solution upon aging, leading to a time-evolved dispersion composed of isolated chains and nanowhiskers. A corresponding evolution of morphology and molecular orientation in the obtained patterns is observed when assembling P3HT solutions with different aging times. The stripes evolve gradually from slim stripes with fingering instabilities for fresh solution into highly regular, perfect stripes for sufficiently aged solution. In the stripe region, P3HT backbone chains align parallel to the contact line for fresh solution whereas perpendicular for aged solution. The thermodynamic multicomponent system gives greater variety to the CESA study.

[Application of regionalized critical neonatal emergency transport system].

OBJECTIVE: To study the application of the regional critical neonatal emergency transport system (NETS) to provide evidence for the optimization of NETS in Beijing. METHODS: All the transported neonates in four hospitals in Haidian District, Beijing, between January 2009 and September 2010 were enrolled. The relevant clinical information of two referral hospitals was analyzed. RESULTS: The top three conditions requiring transport were pre-term delivery, diseases requiring surgical treatment, and respiratory diseases, which accounted for 33.1%, 18.3%, and 14.8%, respectively. Active transport was performed in 95 cases (66.9%) and passive transport in 47 cases (33.1%). The age distribution of the neonates requiring transport was as follows: <6 hrs after birth (24.1%); 6-12 hrs (9.3%); 12-24 hrs (25.9%); and >24 hrs (40.8%). The mean time for transport from the hospital to a referral ward by ambulance was 28.0±11.1 minutes. Diseases requiring emergency surgical treatment were the leading cause of death, accounting for 53.8% of total deaths. The mortality rate was not significantly different between the neonates aged <6 hrs and ≥6 hrs groups. CONCLUSIONS: Active transport remains the main transport pattern among these four hospitals. Neonates requiring surgical treatment have a high mortality rate, and thus special attention should be paid to their transport.

Simultaneous Observation of Mouse Cortical and Hippocampal Neural Dynamics under Anesthesia through a Cranial Microprism Window.

The fluorescence microscope has been widely used to explore dynamic processes in vivo in mouse brains, with advantages of a large field-of-view and high spatiotemporal resolution. However, owing to background light and tissue scattering, the single-photon wide-field microscope fails to record dynamic neural activities in the deep brain. To achieve simultaneous imaging of deep-brain regions and the superficial cortex, we combined the extended-field-of-view microscopy previously proposed with a novel prism-based cranial window to provide a longitudinal view. As well as a right-angle microprism for imaging above 1 mm, we also designed a new rectangular-trapezoidal microprism cranial window to extend the depth of observation to 1.5 mm and to reduce brain injury. We validated our method with structural imaging of microglia cells in the superficial cortex and deep-brain regions. We also recorded neuronal activity from the mouse brains in awake and anesthesitized states. The results highlight the great potential of our methods for simultaneous dynamic imaging in the superficial and deep layers of mouse brains.

The burst of electrophysiological signals in the suprachiasmatic nucleus of mouse during the arousal detected by microelectrode arrays.

The neural mechanisms of torpor have essential reference significance for medical methods and long-term manned space. Changes in electrophysiology of suprachiasmatic nucleus (SCN) conduce to revealing the neural mechanisms from the torpor to arousal. Due to the lower physiology state during the torpor, it is a challenge to detect neural activities in vivo on freely behaving mice. Here, we introduced a multichannel microelectrode array (MEA) for real-time detection of local field potential (LFP) and action potential (spike) in the SCN in induced torpor mice. Meanwhile, core body temperature and behaviors of mice were recorded for further analysis. Platinum nanoparticles (PtNPs) and Nafion membrane modified MEA has a lower impedance (16.58 ± 3.93 kΩ) and higher signal-to-noise ratio (S/N = 6.1). We found that from torpor to arousal, the proportion of theta frequency bands of LFPs increased, spike firing rates rapidly increased. These results could all be characteristic information of arousal, supported by the microscopic neural activity promoting arousal in mice. MEA displayed real-time dynamic changes of neuronal activities in the SCN, which was more helpful to analyze and understand neural mechanisms of torpor and arousal. Our study provided a factual basis for the neural state in SCN of induced non-hibernating animals, which was helpful for the application of clinics and spaceflight.

Self-assembly of virus particles on flat surfaces via controlled evaporation.

Dynamic self-assembly of nonvolatile solutes via controlled solvent evaporation has been exploited as a simple route to create a variety of hierarchically assembled structures. In this work, two glass slides were used to form a confined space in which a solution of a rodlike nanoparticle, tobacco mosaic virus (TMV), was evaporated to create large-scale stripe patterns. The height and width of the stripes are dependent on the TMV concentration. The large-scale-patterned surfaces can be applied to control surface hydrophobicity and direct the growth of bone marrow stromal cells. We systematically studied the effects of stripe width and height on surface hydrophobicity using optical microscopy, atomic force microscopy, and contact angle measurements. This technique offers a facile approach to form 2D patterns on a large surface from a wide range of proteins as well as other biomacromolecules.

Real-time brain-wide multi-planar microscopy for simultaneous cortex and hippocampus imaging at the cellular resolution in mice.

Interactions between the cerebral cortex and the deep cerebellar nuclei play important roles in cognitive processes. However, conventional microscopes fail to dynamically record cellular structures in distinct brain regions and at different depths, which requires high resolution, large field of view (FOV), and depth of field (DOF). Here we propose a single-photon excited fluorescence microscopy technique that performs simultaneous cortex and hippocampus imaging, enabled by a customized microscope and a chronic optical window. After we implant a glass microwindow above the hippocampus, the surface of the hippocampus is shifted to the superficial plane. We demonstrate that the proposed technique is able to image cellular structures and blood vessel dynamics in the cortex and the hippocampus in in vivo experiments, and is compatible with various mesoscopic systems.

Cellular-Scale Microelectrode Arrays to Monitor Movement-Related Neuron Activities in the Epileptic Hippocampus of Awake Mice.

OBJECTIVE: Epilepsy affects 50 million people worldwide and its pathogenesis is still unknown. In particular, the movement-related neural activities involving glutamate (Glu) and electrophysiological signals at cellular level remains unclear. METHODS: A cellular-scale implantable microelectrode array (MEA) was fabricated to detect the movement-related neural activities involving Glu concentration and electrophysiological signals. Platinum and reduced graphene oxide nanocomposites were deposited to enhance the surface area. Glu oxidase (Gluox) were coated to effectively recognize Glu molecule. RESULTS: Neural activities in the hippocampus of normal and epileptic mice is different, and the changes are closely connected with movement. Glu concentration and spike firing rate in the epileptic mice were much higher than those in the normal ones. And the neural activities with significant synchronization were detected in the epileptic mice even without seizure occurrence. Meanwhile, the spikes fire more intensively and Glu level became much higher during the movement of the mice compared to the stationary state. CONCLUSION: The existing abnormality of neural activities in the epileptic mice are potential factors to induce a seizure. Movement may impact the neural activities and the duration of seizure. SIGNIFICANCE: The MEA can monitor changes of movement, Glu and neuron discharges synchronously and provides us an effective technology to understand the neuronal disease.

New electrochemical method for programmed death-ligand 1 detection based on a paper-based microfluidic aptasensor.

As programmed death-ligand 1 (PD-L1) is considered a referenced therapeutic biomarker, a rapid and low-cost method to detect PD-L1 in body fluids is necessary. In this work, a paper-based microfluidic aptasensor for label-free electrochemical detection of PD-L1 in liquids was fabricated. The aptasensor integrates a reaction cell and a three-electrode system, and a differential pulse voltammetry electrochemical method was adopted. PD-L1 aptamer with a low equilibrium dissociation constant was used as a biorecognition molecule. To bind the aptamer and assist in the electrochemical measurement, nanocomposites were synthesized and used to modify the working electrode, which was composed of an amine-functionalized single-walled carbon nanotube, new methylene blue and gold nanoparticles. The basic performance of the aptasensor was tested in phosphate-buffered saline: the linear range was between 10 pg mL-1 and 2.5 ng mL-1, and the detection limit was 10 pg mL-1 (signal/noise = 3). Moreover, the aptasensor was used for the detection of serum samples and compared with an enzyme linked immunosorbent assay (ELISA). The results showed that the aptasensor provides a new low-cost, portable and highly sensitive detection method for PD-L1, as an alternative to ELISA.