This PDF file contains the front matter associated with SPIE Proceedings Volume 10865, including the Title Page, Copyright Information, Table of Contents, Author and Conference Committee lists.

We combined NIR-II illumination at ~1.7 μm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with high spatial resolution in adult mouse brain in vivo, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added as an additional channel to any laser-scanning microscope with low-cost sources and detectors, such as continuous-wave (CW) diode lasers and InGaAs photodiodes. The technique is label-free, simple and requires low illumination power, potentially creating new opportunities for deep tissue imaging in various biological and clinical applications.

In neuroscience, it’s one of the central goals to decipher neuronal ensembles’ function in the neural circuits. Recently, the progress of probes for optogenetic actuators and calcium indicators afford the probability for simultaneous optical manipulation and imaging of neuronal ensembles. Although with point-scanning imaging and localized optical stimulation, previous work had achieved all-optical interrogation of neural circuits in the local brain areas of mice and zebrafish larvae, no attempt was made to optical interrogate neuronal ensembles’ function across the whole-brain. Here, we combined the fast volumetric imaging technique, light-sheet microscopy which was an order of magnitude faster than the point-scanning method, with digital laser beam shaping device, digital micromirror device (DMD), to learn about neuronal activities across the whole brain of behaving larval zebrafish. Using the spectrally separated calcium indicator, GCaMP6f, and activity actuator, ChrimsonR, we can simultaneously image and manipulate neuronal ensembles with minimal spectral cross-talk. Besides, to monitor the behavior of zebrafish larvae, a high-speed camera was introduced into the system. To demonstrate the system’s function, arbitrarily selected neurons was stimulated during the whole-brain neuronal activities’ imaging, which effectively activated the synaptic connections and brain-wide downstream neural circuits and evoked stereotyped behaviors. These results demonstrate the system’s ability in studying the function of brain-wide neuronal ensembles in small animals.

Lattice light sheet (LLS) fluorescence microscopy is a powerful recent technique for in vivo imaging of single and multicellular samples at very high spatio-temporal resolutions. We built a LLS microscope in which we added a photostimulation path to perform all-optical neurophysiological studies in rodent hippocampal brain slices. Thanks to the photo-stimulation path we could achieve fluorescence recovery after photobleaching (FRAP) or glutamate uncaging at spatially and temporally controlled regions of interest. Several fluorescence labelling protocols were employed depending on the imaged structure. Sub-micrometric neuronal elements such as spines or dendritic vesicles could be imaged down to ~20 μm below the surface. We demonstrate the performances of LLS in several ongoing studies: measurement of AMPA receptor surface diffusion at single spines, vesicular transport in dendrites, spontaneous and stimulated local calcium activity in neurons and astrocytes.

Optical brain imaging based on intrinsic signals has revealed new insights into functional brain activation imaging. The main obstacle to this approach is the highly scattering cranial bone over the cortex which hinders the observation of intrinsic optical signals. We have introduced a novel solution for this limitation by proposing a transparent cranial implant providing long-term optical access to the brain, which we call the Window to the Brain (WttB) implant. In this study, we evaluated the feasibility of the WttB implant for multi-wavelength intrinsic optical signal imaging of the brain.

We present results for frequency domain (FD) functional Near Infrared Spectroscopy (fNIRS), where signals from the haemodynamic changes due to focal activations are detected using FD data as collected during a standard retinotopy visual stimulus paradigm. The FD system utilizes radio frequency modulated light to measure phase shift, as well as intensity attenuation, allowing the utilization of two sets of complimentary data for brain activity detection. The information content from measurements of both amplitude and phase, afforded by FD-fNIRS are presented and it is shown that utilization of phase measurement contains unique information regarding both focal activation as well as systematic responses, which could lead to signal suppression from the superficial tissues.

In this study, concurrent electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) recordings were performed based on a modified concealed information test (CIT) task to examine the relationship between the hemodynamic signal of the frontal cortex and event-related potential (ERP) component of P300 for high-sensitivity deception detection. In particular, both the fNIRS data and ERP component of P300 were carefully inspected for participants from both the guilty group and innocent groups. During the performance of CIT task, a series of names were presented, which served as the target, irrelevant, or the probe stimuli for the two groups. The guilty participant who assumed himself (herself) as a spy was instructed to deny the recognition of probe (his (her) own name).Interestingly, we discovered that for the guilty group, the probe stimuli elicited significantly larger P300 at parietal site and also evoked significantly higher HbO concentration changes in bilateral superior frontal gyrus and bilateral middle frontal gyrus than the irrelevants stimuli. However, this is not the case for the innocent group, in which participants didn’t exhibit significant difference in both ERP and fNIRS recordings between the probe and irrelevants stimuli cases. More importantly, our findings also indicated that the combined ERP and fNIRS signals can distinguish well between the guilty and innocent groups, in which AUC (the area under Receiver Operating Characteristic curve) is 0.91 for deception detection based on the combined indicator, much higher than that based on ERP components P300 (0.85) or fNIRS signals (0.84).

Interferometric near-infrared spectroscopy (iNIRS) is a time-of-flight- (TOF-) resolved sensing method for direct and simultaneous quantification of tissue optical properties (absorption and reduced scattering) and dynamics (blood flow index) in vivo with a single modality. The technique has previously been validated in Intralipid phantoms, and applied to continuously and non-invasively monitor optical properties and blood flow index in the brains of head-fixed, anesthetized mice. A demonstration of robust iNIRS measurements in human tissues with motion would support the viability of iNIRS for clinical applications. Here, we perform non-contact iNIRS in human tissues. We show that phase drift caused by involuntary motion during acquisition significantly distorts the optical field autocorrelation, particularly at early TOFs. To solve this issue, we present a novel numerical phase drift correction method to isolate field dynamics due to just red blood cell motion within the sample. Upon correction, TOF-resolved autocorrelations exhibit exponential decay behavior, whether acquired from Intralipid, the human forearm, or the human forehead. We confirm the link between bulk motion artifacts and phase drift by simultaneous, co-registered iNIRS and Optical Coherence Tomography measurements. By applying conventional, time-resolved diffusion theory and diffusing wave spectroscopy theory, we quantify optical properties and time-of-flight-resolved dynamics in Intralipid, the human forearm, and the human brain. Finally, we explore strategies for increased photon collection through parallelization of iNIRS, to probe greater depths in the human brain. This work conclusively shows that diffuse optical measurements of field dynamics are possible, even in the presence of motion artifacts.

High-speed volumetric imaging of neural activity at cellular resolution is important to capture the emergent functional properties of neural circuits. While two-photon calcium imaging provides a power tool to study population activity in vivo, conventional two-photon microscopes only image two-dimensional planes. Expanding it to three-dimensions while maintaining a high spatiotemporal resolution appears necessary. Here, we developed a novel two-photon microscope with dual-color laser excitation that can image neural activity in a 3D volume with high spatiotemporal resolution. We use 920 nm and 1064 nm lasers to image, at the same time, neurons labeled with GCaMP6 in mice cortical layer 2/3, and with jRGECO in layer 5, respectively. An electrically tunable lens or a spatial light modulator was implemented in the beam path to enable fast sequential or simultaneous imaging of different focal planes. Using this beam multiplexing strategy, we image the neuronal activity of cortical circuits at high speed in primary visual cortex from awake mice (from layer 1 to 5 at 10 vol/s). We analyze the orientation tuning properties of cells in cortical columns, as well as the spatial structures of visually-evoked neuronal ensembles. Furthermore, we demonstrate fast volumetric calcium imaging of layer 1 apical dendrites and layer 2/3 somata in local V1 circuits, as well as long-range projections from PFC and layer 2/3 somata in V1.

Two-photon microscopy is a powerful tool for in vivo brain imaging that has greatly facilitated the neuroscience research in the past few decades. However, it still remains a challenge to image deep inside the brain with near diffraction-limited resolution due to the optical aberrations induced by the biological tissue and the cranial window. Here, we used an adaptive optics approach based on direct wavefront sensing to correct the aberration induced by the thinned skull window and achieved minimally invasive imaging of cerebral cortex with near-diffraction-limit resolution. Besides, by compensating the intrinsic aberration of a miniature gradient-index lens that implanted into the brain, two-photon imaging of hippocampal dendritic spines was realized over an extended field of view. The improvement in fluorescence intensity and imaging resolution enabled us to resolve the fine structures in live mouse brain such as dendritic spines that were invisible without the help of adaptive optics.

In the brain, neurotransmitters or neuromodulators play pivotal roles in chemical synaptic transmission and consequently, monitoring their dynamics, especially in vivo, is critical for understanding their physiological- or pathophysiological roles at molecular, cellular, and circuit levels during behaviors and/or during diseases. We recently developed genetically-encoded GPCR-activation based (GRAB) sensors capable of reporting dynamics of acetylcholine, dopamine and norepinephrine with rapid kinetics, chemical- and cell-specificity in multiple organisms in vivo. Here, we explored the usage of G protein derivatives, either mini-G proteins or C-terminal peptides of Gα subunit to engineer new GRAB sensors. We found that the conformational changes mediated by mini-G proteins interacting with GPCRs, or Gα Cterminal peptides interacting with GPCRs could be harnessed to regulate fluorescence outputs of a GPCR fused circular permuted GFP (cpGFP). In addition, inter-molecular fusion of Gα C-terminal peptides significantly suppressed ectopic activation of G protein signaling in a GRAB acetylcholine sensor. Finally, we showed Gα C-terminal peptides fusion strategy could be applied to generate various GRAB sensors for small molecular compounds or neuropeptides.

Neuro-rehabilitative research is developing novel strategies to enhance the effectiveness of therapies after stroke by using a combination of physical and plasticizing treatments ^1-3. Previous studies have shown that repeated optogenetics stimulation of neurons in the peri-lesioned area induces a significant improvement in cerebral blood flow and neurovascular coupling response ^4-6. Up to now the mechanisms underneath the reshaping of brain circuitry induced by rehabilitation after stroke are widely unknown. To investigate how rehabilitative therapies shape new cortical maps in the peri-infarct region, we induce a photothrombotic stroke in the primary motor cortex and the expression of Channelrhodopsine 2 (ChR2) in the peri-infarct area on Thy1-GCaMP6f mice. To promote functional recovery after stroke we use both an optogenetic strategy to stimulate targeted excitatory neurons in the peri-lesional region and motor training on a robotic platform (M-Platform) ⁷. A 473 nm laser repeatedly stimulates ChR2-transfected neurons; the optostimulation is performed five days a week. The motor rehabilitation consists in a pulling task: after the forelimb is passively extended by the linear actuator of the M-platform, the animal has to pull back up to the resting position. By analysing the spatio-temporal calcium dynamic and the reshaping of cortical activation area during the movement throughout the treatment period, we found that the combined treatment restores cortical activation profiles during the forelimb movement. Through behavioural experiments, using Schallert test, we also evaluate changes of forelimb functionality during rehabilitation. Our combination of techniques allows obtaining unprecedented views on cortical plasticity induced by rehabilitative therapies.

Here we present preliminary experimental data suggesting about involvement of the meningeal and cervical lymphatics in neurorehabilitation. Using model of hemorrhagic stroke, immunohistochemical analysis and atomic absorption spectroscopy, we clearly demonstrate the lymphatic clearance from the blood after stroke via the meningeal lymphatic vessels with further accumulation of hemosiderin and iron (products of disaggregated hemoglobin) in the deep cervical node (dcLN). The optical coherent tomography (OCT) was used for in vivo monitoring of accumulation of gold nanorods (92 nm in diameter) in the dcLN after their injection into the cisterna magna with the aim of mimicking of the brain clearance from of blood. The both ex vivo and in vivo data show the lymphatic clearance from subjects (the blood/GNRs) injected into the subarachnoid space that might be an important mechanism of neurorehabilitation after the intracranial hemorrhages.

The morphological and functional changes in cerebral blood vessels network is not well characterized in mice models of obesity. In order to study the hemodynamics of these models at rest and during sensory stimulation, we have developed a multi exposure speckle imaging system. It allows wide field superficial imaging of blood flow of the mice cortex. We have characterized the performances of the system using microfluidic phantoms. The capacity of the technique to retrieve accurate relative flow values was studied as a function of the diameter of vessels and the scatterers concentration. New biological data have been obtained in mice models of obesity (high fat diet mice) at rest and under sensory activation.

Photoacoustic microscopy (PAM) is an emerging technique extensively used to study brain activities. However, limited by the size and performance of optical/acoustic scanners, existing ORPAMs are still bulky, heavy, and suffer from low imaging quality/speed. Here, we develop a wearable ORPAM probe featuring small, light, fast, and cellular imaging capability. To evaluate the probe, we monitored the cerebral vascular network within two hours and demonstrated that the microscope was stable enough to study long-term brain hemodynamics in a freely moving rat.

In this study, we developed a wide-field all-optical system based on a red-shifted GECI (RCaMP1a) combined with channelrhodopsin II (ChR2) for simultaneous stimulation and readout of neuronal activity. Our results show that RCaMP1a transfection in primary motor cortex extends all over the cortical motor areas. The RCaMP1a and ChR2 reporter expression largely overlap, thus allowing the stimulation and readout from the same functional areas. Furthermore, we characterized the functional response by performing single pulse optogenetic stimulation and we observed that evoked calcium signals increase at increasing laser power. In order to study the cortical activation underlying a specific motor behavior, we performed optogenetic-stimulation of the Rostral Forelimb Area (RFA) with a train of lasers pulses. We observed that during 1s of 16 Hz train stimulus the animals suddenly start grasping with the contralateral forelimb. Cortical dynamics recorded during the optogenetically-triggered motor task show correlated activity between the RFA and the nearby motor areas. The all-optical system optimization and the possibility to link the neuronal population activity with the animal behavior would be a key point in understanding the network activity underlying a specific behavior.

Neural Imaging and Sensing are widely utilized in neuroscience research. The entire technical chain usually includes labeling, imaging, and image processing. Novel techniques are developed in rapid succession, and new applications follows. This presentation will focus on a bibliometrics study related to the three directions of the emerging neural imaging and sensing techniques. Based on Web of Science and Scopus, the hot topics are easily selected. We will highlight several typical techniques emerged in recent years, and discuss their advantages and specialized applications. Among those emerging techniques, a new crossdisciplinary field, brainsmatics, is growing up. Brainsmatics is the shorten term of Brain Spatial Informatics, which develops methods and tools for understanding brain based on brain spatial information. In neuroscience, scientific questions are focused and answered mainly in molecular, cellular, genetic, and electrophysiological levels, respectively. A full understanding of the brain calls for the integration of brain information in all levels. To combine all these different level data, the spatial information is the key reference. High resolution and precision positioning are two challenges in brainsmatics, while the image standard and brain-wide coordinate system definition are also important.

In neuroscience, Brain template images and annotation atlases are key to analyzing whole brain microscopy images in a common coordinate framework (CCF) and sharing of anatomical data among neuroscientists. Mapping individual animal specimen brain to a common space allows neuroscientists compare and utilize data from experiments done on other modalities or markers. With the advent of new clearing and novel light microscopy techniques, large number of samples are imaged in these unique combinations of clearing and microscopy that the experimenter deems best to bring out the best signal-to-noise ratio for their biological data. This paper describes our framework that each such experimenter to create a template brain registered with the Allen CCF for their unique combination. Further, we demonstrate the ability of the framework to create a template brain for an enhanced delipidation protocol called UClear [Unpublished] and imaged using LaVision BioTech Ultramicroscope II. The cleared brains were imaged in dibenzyl ether (DBE) under 488-nm illumination conditions that allowed the visualization of major brain regions based on intrinsic tissue autofluorescence. As contrast in these images differs from brains imaged by serial two-photon tomography used to generate the CCF space of the Allen Mouse Brain Atlas, we developed a new CCF brain template for processing and analysis of the UClear brains. This brain template generation method can make the registration pipeline agnostic to the tissue clearing and imaging protocol.

We propose a miniaturized intrinsic optical sensing system (MiniIOS) for monitoring the hemodynamic response from the brain surface of freely-behaving rodents. It integrates the light source, detector and data acquisition in a printed circuit board with a dimension of 5.5x4.7x1.8 mm³. This paper presents the system design, simulation and preliminary characterization.

One of the major goals in neuroscience is to identify the enormous diversity of neurons, depict their heterogeneity, and explore their function under normal and pathological conditions. To date, the repetitive study of specific neurons in the vertebrate nervous system is almost impossible. Invertebrate nervous systems, on the other hand, have provided an opportunity to study single identified neurons for decades. However, simple methods to study the identified neurons in cell culture are still lacking. In this research study we developed a simple method, based on microinjection, to study and manipulate single identified neurons in culture. The concept behind this method is to utilize the advantages and simplicity of the leech nervous system. Within the leech ganglion, neurons are arranged in a characteristic and stereotypical manner. Their location, size, and biophysical properties are highly characterized. This allows us to identify and label specific cells of interest prior to plating. These cells can be later identified throughout their culture development solely by fluorescent imaging thousands of cells in a cell culture consisting of neuronal and non-neuronal cell populations. Here, we present a proof of concept for this method. We demonstrate neuronal viably by following the procedure up to 7 days and by introducing different molecules to different neurons of interest. This method can easily be adapted to other invertebrate brains and opens up new possibilities for precise manipulation, study, and characterization of specific individual neurons in cell culture.

The physiology of individual synapses on dendritic spines is important because isolated and widely distributed spines are active during sensory information processing in vivo. We used acute cortical brain slices from the rat to investigate synaptic signal integration at the level of individual synapses on dendritic spines in layer 5 pyramidal neurons. We monitored subthreshold synaptic signals using a combination of voltage-sensitive dye recordings, patch-electrode somatic recordings, and 2-photon glutamate uncaging with holographic illumination. We describe the capabilities and limitations of this approach and demonstrate that imaging with an organic voltage-sensitive dye is presently a unique way to monitor temporal summation of uncaging evoked quantal excitatory synaptic potentials locally, at the site of origin on thin basal dendrites. The results indicated that the ability of repetitive activation of individual excitatory synapses on spines to influence the electrical signaling in individual neurons is strictly limited to subthreshold responses. We show that the underlying mechanisms, which control the temporal summation of excitatory synaptic potential in single synapses, can now be investigated with described methodology.

Due to the frequent attacks using improvised explosive devices (IEDs), the number of patients suffering from blast-induced traumatic brain injury (bTBI) has been growing. Although most of the patients have been diagnosed as having mild bTBI, many of them show higher brain dysfunction in the chronic phase. However, the mechanisms of bTBI are unclear, and methods of prevention, diagnosis and treatment have therefore not been established. In our previous study, we applied a laser-induced shock wave (LISW) to the rat brain, for which real-time measurements of cerebral hemodynamics were conducted based on diffuse reflectance spectroscopy. We found that LISW application caused spreading depolarization (SD) and transient hyperemia/hyperoxemia, which was followed by persistent oligemia/hypoxemia in the cortex. We hypothesized that nitric oxide (NO) may be involved in these abnormal hemodynamic changes. In this study, we investigated our hypotheses using an inhibitor of NO synthesis. We observed that by inhibiting NO synthesis with LNAME, transient hyperemia and persistent oligemia/hypoxemia were reduced, suggesting that NO generation is activated by shock wave exposure and causes the abnormal hemodynamic changes in the rat brain.

Three-dimensional mapping of skeletal muscle is particularly valuable for systematical identification and analysis of various biological components, such as nerve fibers, muscle fibers and vessels during disease and regeneration. iDISCO, as a whole-mount immunolabeling technique, provides an important tool for volume imaging of muscles, but its application has been limited to thin and small muscles of young mice owing to poor antibody penetration. In this work, we developed a modified iDISCO method and applied it to label the nerve fibers in various skeletal muscles of adult mice, including diaphragm, biceps brachii and tibialis anterior. Light-sheet microscopy was used to image biceps brachii and tibialis anterior. The results showed that the modified method could achieve uniform and complete labeling of nerve fibers within muscles, whereas original iDISCO method caused strong nonspecific signal in the surface of the muscles, and intramuscular nerve fibers were almost invisible. The modified method permitted us to trace three-dimensional nerve fibers in the muscles. This method shows potential for three-dimensional histological analysis in mouse skeletal muscle, facilitating the understanding of structural-functional relationship of skeletal muscle in physiological and pathological condition.

In this paper, the effect of various channel selection strategies on the initial dip phase of the hemodynamic response (HR) using functional near-infrared spectroscopy (fNIRS) is investigated. The strategies using channel averaging, channel averaging over a local region, t-value-based channel selection, baseline correction, and vector phase analysis are examined. For t-value-based channel selection, three gamma functions are used to model the initial dip, the main HR, and the undershoot in generating the designed HR function. The linear discriminant analysis based classification accuracy is used as performance evaluation criteria. fNIRS signals are obtained from the left motor cortex during righthand thumb and little finger tapping tasks. In classifying two finger tapping tasks, signal mean and minimum value during 0~2.5 sec, as features of initial dip, are used. The results show that the active channel selected using t-value and vector phase analysis yielded the highest averaged classification accuracy. It is also found that the initial dip in the HR disappears in case of averaging overall channels. The results demonstrated the importance of the channel selection in improving the classification accuracy for fNIRS-based brain-computer interface applications. Furthermore, the use of three gamma functions can also be useful for fNIRS brain imaging for detecting the initial dip in the HR.