This PDF file contains the front matter associated with SPIE Proceedings Volume 10051, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

We report a novel use of optical coherence tomography (OCT) based angiography to visualize and quantify dynamic response of cerebral capillary flow pattern in mice upon hindpaw electrical stimulation through the measurement of the capillary transit-time heterogeneity (CTH) and capillary mean transit time (MTT) in a wide dynamic range of a great number of vessels in vivo. The OCT system was developed to have a central wavelength of 1310 nm, a spatial resolution of ~8 µm and a system dynamic range of ~105 dB at an imaging rate of 92 kHz. The mapping of dynamic cerebral microcirculations was enabled by optical microangiography protocol. From the imaging results, the spatial homogenization of capillary velocity (decreased CTH) was observed in the region of interest (ROI) corresponding to the stimulation, along with an increase in the MTT in the ROI to maintain sufficient oxygen exchange within the brain tissue during functional activation. We validated the oxygen consumption due to an increase of the MTT through demonstrating an increase in the deoxygenated hemoglobin (HbR) during the stimulation by the use of laser speckle contrast imaging.

A visible light spectral domain optical coherence microscopy system operating in the wavelength range of 450-680 nm was developed. The resulting large wavelength range of 230 nm enabled an ultrahigh axial resolution of 0.88μm in tissue. The setup consisted of a Michelson interferometer combined with a homemade spectrometer with a spectral resolution of 0.03 nm. Scanning of 1 x 1 mm² and 0.5 x 0.5 mm² areas was performed by an integrated microelectromechanical mirror. After scanning the light beam is focused onto the tissue by a commercial objective with a 10 x magnification, resulting in a transverse resolution of 2 μm . Specification measurements showed that a -89 dB sensitivity with a 24 dB/mm roll-off could be achieved with the system. First of all the capabilities of the system were tested by investigating millimeter paper, tape and the USAF (US Air Force) 1951 resolution test target. Finally cerebral tissues from non-pathological and Alzheimer's disease affected brains were investigated. The results showed that structures, such as white and gray matter, could be distinguished. Furthermore a first effort was made to differentiate Alzheimer's disease from healthy brain tissue.

The Western diet, causative in the development of atherosclerotic cardiovascular disease, has recently been associated with the development of diffuse white matter disease (WMD) and other subcortical changes. Yet, little is known about the pathophysiological mechanisms by which a high-fat diet can cause WMD. Mechanistic studies of deep brain regions in mice have been challenging due to a lack of non-invasive, high-resolution, and deep imaging technologies. Here we used Optical Coherence Tomography to study mouse cortical/subcortical structures noninvasively and in vivo. To better understand the role of Western Diet in the development of WMD, intensity and Doppler flow OCT images, obtained using a 1300 nm spectral / Fourier domain OCT system, were used to observe the structural and functional alterations in the cortex and corpus callosum of Western Diet and control diet mouse models. Specifically, we applied segmentation to the OCT images to identify the boundaries of the cortex/corpus callosum, and further quantify the layer thicknesses across animals between the two diet groups. Furthermore, microvasculature alterations such as changes in spatiotemporal flow profiles within diving arterioles, arteriole diameter, and collateral tortuosity were analyzed. In the current study, while the arteriole vessel diameters between the two diet groups was comparable, we show that collateral tortuosity was significantly higher in the Western diet group, compared to control diet group, possibly indicating remodeling of brain vasculature due to dietary changes. Moreover, there is evidence showing that the corpus callosum is thinner in Western diet mice, indicative of tissue atrophy.

We present the serial optical coherence scanner (SOCS), which consists of a polarization sensitive optical coherence tomography and a vibratome with associated controls for serial imaging, to visualize the cerebellum and adjacent brainstem of mouse. The cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. Images from serial scans reveal the large-scale anatomy in detail and map the nerve fiber pathways in the cerebellum and adjacent brainstem. The optical system, which has ~5.5 μm axial resolution, utilizes a scan lens or a water-immersion microscope objective resulting in 10 μm or 4 μm lateral resolution, respectively. The large-scale brain imaging at high resolution requires an efficient way to collect large datasets. It is important to improve the SOCS system to deal with large-scale and large number of samples in a reasonable time. The imaging and slicing procedure for a section took about 4 minutes due to a low speed of the vibratome blade to maintain slicing quality. SOCS has potential to investigate pathological changes and monitor the effects of therapeutic drugs in cerebellar diseases such as spinocerebellar ataxia 1 (SCA1). The SCA1 is a neurodegenerative disease characterized by atrophy and eventual loss of Purkinje cells from the cerebellar cortex, and the optical contrasts provided by SOCS is being evaluated for biomarkers of the disease.

Brain tumors are characterized with morphological changes at cellular level such as enlarged, non-spherical nuclei, microcalcifications, cysts, etc., and are highly vascularized. In this study, two research-grade optical coherence tomography (OCT) systems operating at ~800 nm and ~1060 nm with axial resolution of 0.95 µm and 3.5 µm in biological tissue respectively, were used to image in vivo and ex vivo the structure of brain tumours in rats. Female Fischer 344 rats were used for this study, which has received ethics clearance by the Animal Research Ethics Committees of the University of Waterloo and the University Health Network, Toronto. Brain tumours were induced by injection of rat brain cancer cell line (RG2 glioma) through a small craniotomy. Presence of brain tumours was verified by MRI imaging on day 7 post tumour cells injection. The in vivo OCT imaging session was conducted on day 14 of the study with the 1060 nm OCT system and both morphological OCT, Doppler OCT and OMAG images were acquired from the brain tumour and the surrounding healthy brain tissue. After completion of the imaging procedure, the brains were harvested, fixed in formalin and reimaged after 2 weeks with the 800 nm OCT system. The in vivo and ex vivo OCT morphological images were correlated with H and E histology. Results from this study demonstrate that UHR-OCT can distinguish between healthy and cancerous brain tissue based on differences in structural and vascular pattern.

Microelectrodes implanted in the brain cause mechanical damage to the tissue that mediate neuroinflammation and eventual encapsulation by microglia and astrocytes. Electrophysiological signals recorded from implants used in brain-computer interfaces (BCI) degrade over time, limiting their usefulness, but the precise causes and progression are not fully understood. We are investigating the dynamics of brain morphological changes and neuroinflammation with a multimodal approach to better understand the potential causes of implant failure. We performed weekly optical coherence tomography (OCT)-guided two-photon microscopy (TPM) in the region around microelectrodes inserted under a cranial window concurrent with electrophysiological recordings. Transgenic mouse cohorts studied include Thy1-YFP, Cx3cr1, and GFAP-GFP to image neurons, microglia, and astrocytes, respectively. Single-shank, 16-channel, Michigan-style microelectrodes were inserted under the window at a 15-20° angle with an insertion depth up to cortical layer 5. Single-unit and local field potential (LFP) recordings were collected for 15 minutes while the animals moved freely in their home cages. Cellular and vascular morphology were monitored using TPM and OCT at timepoints matched to the recordings. In preliminary data, we observed a decay of neural firing rates in most of the channels after implantation. The relationship between electrophysiological measures (e.g., neural firing rate, LFP power) and neural/vascular morphological measurements (e.g., cell density, glial migration, blood flow changes) will be quantified. The multimodal approach combining electrophysiology and optical imaging provides a broader picture of the multifactorial nature of the response to implanted electrodes. Understanding and accounting for the response may lead to better BCI designs and approaches.

Epilepsy is a chronic neurological disorder characterized by recurrent and unpredictable seizures. Electrophysiology has remained the gold standard of neural activity detection but its resolution and high susceptibility to noise and motion artifact limit its efficiency. Optical imaging techniques, including fMRI, intrinsic optical imaging, and diffuse optical imaging, have also been used to detect neural activity yet these techniques rely on the indirect measurement of changes in blood flow. A more direct optical imaging technique is optical coherence tomography (OCT), a label-free, high resolution, and minimally invasive imaging technique that can produce depth-resolved cross-sectional and 3D images. In this study, OCT was used to detect non-vascular depth-dependent optical changes in cortical tissue during 4-aminopyridine (4-AP) induced seizure onset. Calculations of localized optical attenuation coefficient (µ) allow for the assessment of depth-resolved volumetric optical changes in seizure induced cortical tissue. By utilizing the depth-dependency of the attenuation coefficient, we demonstrate the ability to locate and remove the optical effects of vasculature within the upper regions of the cortex on the attenuation calculations of cortical tissue in vivo. The results of this study reveal a significant depth-dependent decrease in attenuation coefficient of nonvascular cortical tissue both ex vivo and in vivo. Regions exhibiting decreased attenuation coefficient show significant temporal correlation to regions of increased electrical activity during seizure onset and progression. This study allows for a more thorough and biologically relevant analysis of the optical signature of seizure activity in vivo using OCT.

The combination of optogenetics and optical imaging modalities has become a popular tool for the investigation of neurovascular coupling. Developing a closed-loop hemodynamic control system capable of dynamically following various blood flow patterns could be beneficial to the causal investigation of neurovascular coupling. To develop this closed-loop hemodynamic control system, we have added a compensator to create a loop consisting of optogenetic stimulation, neural activities, neurovascular coupling, the evoked hemodynamic response, and a blood flow monitoring device to continuously minimize the difference between the recorded blood flow values and desired blood flow patterns. A Real-time Doppler Optical Coherence Tomography (D-OCT) is employed in this study to monitor the cross-sectional velocity profile of a vessel at a frame rate of 71 frames per second. At the same time, a proportional-derivative compensator is used to continuously adjust the pulse width of a 450nm pulsed laser that delivers 15 Hz photostimulation to the cerebral cortex of Thy1-Channelrhodopsin-2 mice. For each vessel, time-varying desired patterns and stimulation parameters were chosen according to the effect of pulse width modulation on its hemodynamic response, then proportional and derivative gains were tuned to produce a near-critically damped response. After parameter optimization, the closed-loop hemodynamic compensator successfully controlled the blood flow in middle cerebral artery branches.

No current imaging technology allows us to directly and without significant distortion visualize the microscopic and defining anatomical features of the human brain. Ex vivo histological techniques can yield exquisite planar images, but the cutting, mounting and staining that are required components of this type of imaging induce distortions that are different for each slice, introducing cross-slice differences that prohibit true 3D analysis. We are overcoming this issue by utilizing Optical Coherence Tomography (OCT) with the goal to image whole human brain cytoarchitectural and laminar properties with potentially 3.5 µm resolution in block-face without the need for exogenous staining. From the intrinsic scattering contrast of the brain tissue, OCT gives us images that are comparable to Nissl stains, but without the distortions introduced in standard histology as the OCT images are acquired from the block face prior to slicing and thus without the need for subsequent staining and mounting. We have shown that laminar and cytoarchitectural properties of the brain can be characterized with OCT just as well as with Nissl staining. We will present our recent advances to improve the axial resolution while maintaining contrast; improvements afforded by speckle reduction procedures; and efforts to obtain quantitative maps of the optical scattering coefficient, an intrinsic property of the tissue.

Deciphering the fine morphology and precise location of neurons and neural circuits are crucial to enhance our understanding of brain function and diseases. Traditionally, we have to map brain images to coarse axial-sampling planar reference atlases to orient neural structures. However, this means might fail to orient neural projections at single-cell resolution due to position errors resulting from individual differences at the cellular level. Here, we present a high-throughput imaging method that can automatically obtain the fine morphologies and precise locations of both neurons and circuits, employing wide-field large-volume tomography to acquire three-dimensional images of thick tissue and implementing real-time soma counterstaining to obtain cytoarchitectonic landmarks during the imaging process. The reconstruction and orientation of brain-wide neural circuits at single-neuron resolution can be accomplished for the same mouse brain without additional counterstains or image registration. Using our method, mouse brain imaging datasets of multiple type-specific neurons and circuits were successfully acquired, demonstrating the versatility. The results show that the simultaneous acquisition of labeled neural structures and cytoarchitecture reference at single-neuron resolution in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. Our method provides a novel and effective tool for application in studies on genetic dissection, brain function and the pathology of the nervous system.

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6. Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity. We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.

We have fabricated sub-micron opto-chemical probes for pH, oxygen and calcium monitoring and demonstrated their application in intracellular and extracellular monitoring of neurons (cortical neuronal cultures and acute hippocampal slices). Using these probes, we have measured extracellular pH in the stratum radiatum of the CA1 region of mouse hippocampus upon stimulation of presynaptic Schaffer collateral axons. Synaptic transmission was monitored using standard electrophysiological techniques. We find that the local pH transiently changes in response to synaptic stimulation. In addition, the geometry of the functionalized region on the probe combined with high sensitivity imaging enables simultaneous monitoring of spatially adjacent but distinct compartments. As proof of concept we impaled cultured neurons with the probe measured calcium and pH inside as well as directly outside of neurons as we changed the pH and calcium concentration in the physiological solution in the perfusion chamber. As such these probes can be used to study the impact of the environment on both cellular and extra-cellular space. Additionally as the chemical properties of the surrounding medium can be controlled and monitored with high precision, these probes enable differential measurement of the target parameter referenced to a stable bath. This approach eliminates the uncertainties associated with non-chemical fluctuations in the fluorescent emission and result in a self-calibrated opto-chemical probe. We have also demonstrated multifunctional probes that are capable of measuring up to three parameters in the extracellular space in brain slices.

We developed a novel reactive embedding resin that crosslinking with the biological tissue via the reaction of epoxy group and amino group, which improves its compatibility with biological tissue and can be good to preserve endogenous fluorescent protein and dyes.

Chronic hypertension itself does not cause stroke but significantly decreases the resistant to stroke induced by stress due to exhausting of adaptive capacity of cerebral endothelium and decrease resistance of blood-brain barrier to stress.

The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.

Adverse childhood experiences have lasting detrimental effects on mental health and are strongly associated with impaired cognition and increased risk of developing psychopathologies. Preclinical and neuroimaging studies have suggested that traumatic events during brain development can affect cerebral myelination particularly in areas and tracts implicated in mood and emotion. Although current neuroimaging techniques are quite powerful, they lack the resolution to infer myelin integrity at the cellular level. Recently demonstrated coherent Raman microscopy has accomplished cellular level imaging of myelin sheaths in the nervous system. However, a quantitative morphometric analysis of nerve fibers still remains a challenge. In particular, in brain, where fibres exhibit small diameters and varying local orientation. In this work, we developed an automated myelin identification and analysis method that is capable of providing a complete picture of axonal myelination and morphology in brain samples. This method performs three main procedures 1) detects molecular anisotropy of membrane phospholipids based on polarization resolved coherent Raman microscopy, 2) identifies regions of different molecular organization, 3) calculates morphometric features of myelinated axons (e.g. myelin thickness, g-ratio). We applied this method to monitor white matter areas from suicides adults that suffered from early live adversity and depression compared to depressed suicides adults and psychiatrically healthy controls. We demonstrate that our method allows for the rapid acquisition and automated analysis of neuronal networks morphology and myelination. This is especially useful for clinical and comparative studies, and may greatly enhance the understanding of processes underlying the neurobiological and psychopathological consequences of child abuse.

Neurorehabilitation protocols based on the use of robotic devices provide a highly repeatable therapy and have recently shown promising clinical results. Little is known about how rehabilitation molds the brain to promote motor recovery of the affected limb. We used a custom-made robotic platform that provides quantitative assessment of forelimb function in a retraction test. Complementary imaging techniques allowed us to access to the multiple facets of robotic rehabilitation-induced cortical plasticity after unilateral photothrombotic stroke in mice Primary Motor Cortex (Caudal Forelimb Area - CFA). First, we analyzed structural features of vasculature and dendritic reshaping in the peri-infarct area with two-photon fluorescence microscopy. Longitudinal analysis of dendritic branches and spines of pyramidal neurons suggests that robotic rehabilitation promotes the stabilization of peri-infarct cortical excitatory circuits, which is not accompanied by consistent vascular reorganization towards pre-stroke conditions. To investigate if this structural stabilization was linked to functional remapping, we performed mesoscale wide-field imaging on GCaMP6 mice while performing the motor task on the robotic platform. We revealed temporal and spatial features of the motor-triggered cortical activation, shining new light on rehabilitation-induced functional remapping of the ipsilesional cortex. Finally, by using an all-optical approach that combines optogenetic activation of the contralesional hemisphere and wide-field functional imaging of peri-infarct area, we dissected the effect of robotic rehabilitation on inter-hemispheric cortico-cortical connectivity.

We describe a scanning near-infrared fluorescence imager for through-skull non-invasive brain imaging on live murine models. The captured photoluminescence feature through scattering media was enhanced using a high sensitivity scientific CMOS sensor with the obtained spatial resolution of 15.63 μm, depth of field of 5 mm and an average local signal-to-noise ratio of 37.5 dB.

Gradual changes in brain microvasculature and cerebral capillary blood flow occurring with atherosclerosis may significantly contribute to cognition decline due to their role in brain tissue oxygenation. However, previous stud- ies of the relationship between cerebral capillary blood flow and brain tissue oxygenation are limited. This study aimed to investigate vascular and concomitant changes in brain tissue pO₂ with atherosclerosis. Experiments in young healthy C57B1/6 mice (n=6 , WT), young atherosclerotic mice (n=6 , ATX Y) and old atherosclerotic mice (n=6 , ATX O) were performed imaging on the left sensory-motor cortex at resting state under urethane (1.5 g/kg) anesthesia using two-photon fluorescence microscopy. The results showed that pO₂ around capillaries, correlated with red blood cell (RBC) flux, increased with atherosclerosis.

Gliomas are known to cause significant changes in normal brain function that lead to cognitive deficits. Disruptions in resting state networks (RSNs) are thought to underlie these changes. However, investigating the effects of glioma growth on RSNs in humans is complicated by the heterogeneity in lesion size, type, and location across subjects. In this study, we evaluated the effects of tumor growth on RSNs over time in a controlled mouse model of glioma growth. Methods: Glioma cells (5x104-105 U87s) were stereotactically injected into the forepaw somatosensory cortex of adult nude mice (n=5). Disruptions in RSNs were evaluated weekly with functional connectivity optical intrinsic signal imaging (fcOIS). Tumor growth was monitored with MRI and weekly bioluminescence imaging (BLI). In order to characterize how tumor growth affected different RSNs over time, we calculated a number of functional connectivity (fc) metrics, including homotopic (bilateral) connectivity, spatial similarity, and node degree. Results: Deficits in fc initiate near the lesion, and over a period of several weeks, extend more globally. The reductions in spatial similarity were found to strongly correlate with the BLI signal indicating that increased tumor size is associated with increased RSN disruption. Conclusions: We have shown that fcOIS is capable of detecting alterations in mouse RSNs due to brain tumor growth. A better understanding of how RSN disruption contributes to the development of cognitive deficits in brain tumor patients may lead to better patient risk stratification and consequently improved cognitive outcomes.

The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. Measurements of CMRO2 with high spatio-temporal resolution are critically important for understanding how the brain copes with metabolic and blood perfusion changes associated with various clinical conditions, such as stroke, periinfarct depolarizations, and various microvasculopathies (e.g., Alzheimer’s disease, chronic hypertension). CMRO2 measurements are also important for understanding the physiological underpinnings of functional Magnetic Resonance Imaging signals. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a novel method that allows estimation of CMRO2 based on a single measurement modality - two-photon phosphorescence lifetime microscopy (2PLM) imaging of the partial pressure of oxygen (PO2) in cortical tissue. CMRO2 is estimated by fitting the changes of tissue PO2 around cortical penetrating arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats, and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method has a spatial resolution of approximately 200 μm and it may provide CMRO2 measurements in individual cortical layers or within confined cortical regions such as in ischemic penumbra and the foci of functional activation.

We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

We characterized cortical microvascular PO2 and blood flow changes in response to whisker stimulation in awake mice. The measurements were performed by combining two-photon microscopy imaging of the cortical oxygenation and optical coherence tomography imaging of the cerebral blood flow. In order to perform fast spatio-temporally resolved measurements of PO2, we used a newly-developed oxygen-sensitive probe PtG-2P, which has significantly higher brightness than the established two-photon-enhanced oxygen sensor PtP-C343. We characterized the performance of the new probe in vivo and mapped the amplitudes and shapes (e.g. initial dip, overshoot, and post stimulus undershoot) of the PO2 changes as a function of the vessel type (e.g., arterioles, capillaries, and venules) and a distance from the activation center. The measurements in the awake mice are not affected by the confounding factors of anesthesia on the animal physiology, including the level of cerebral metabolism and the amplitude and speed of neuronal and vascular responses. Our results will help to understand changes in oxygenation and blood flow on the cortical microvascular scale, will lead to improved understanding of the cerebral physiology, pathophysiology and will improve quantitative interpretation of fMRI signals.

Light sheet fluorescent microscopy is able to provide high acquisition speed and high contrast images, as well as the low photo-bleaching and photo-damage brought to the sample. Here we describe a compact setup design optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We report this prototype instrument is capable of rapid imaging wide area of the dendritic or axonal arbor of a dye-filled neuron in hippocampal slice. We also show several applications of this compact light sheet fluorescent microscope, to demonstrate that our approach offers a powerful functionality to the neuroscience community that is not achievable with traditional imaging methods.

Infrared neural stimulation (INS) provides optical control of neural excitability using near to mid-infrared (mid-IR) light, which allows for spatially selective, artifact-free excitation without the introduction of exogenous agents or genetic modification. Although neural excitability is mediated by a transient temperature increase due to water absorption of IR energy, the molecular nature of IR excitability in neural tissue remains unknown. Current research suggests that transient changes in local tissue temperature give rise to a myriad of cellular responses that have been individually attributed to IR mediated excitability. To further elucidate the underlying biophysical mechanisms, we have begun work towards employing a novel multimodal nonlinear imaging platform to probe the molecular underpinnings of INS. Our imaging system performs coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and thermal imaging into a single platform that allows for unprecedented co-registration of thermal and biochemical information in real-time. Here, we present our work leveraging CARS and SRS in acute thalamocortical brain slice preparations. We observe the evolution of lipid and protein-specific Raman bands during INS and electrically evoked activity in real-time. Combined with two-photon fluorescence and second harmonic generation, we offer insight to cellular metabolism and membrane dynamics during INS. Thermal imaging allows for the coregistration of acquired biochemical information with temperature information. Our work previews the versatility and capabilities of coherent Raman imaging combined with multiphoton imaging to observe biophysical phenomena for neuroscience applications.

General anesthetics are known to have profound effects on cerebral hemodynamics and neuronal activities. However, it remains a challenge to directly assess anesthetics-induced hemodynamic and oxygen-metabolic changes from the true baseline under wakefulness at the microscopic level, due to the lack of an enabling technology for high-resolution functional imaging of the awake mouse brain. To address this challenge, we have developed head-restrained photoacoustic microscopy (PAM), which enables simultaneous imaging of the cerebrovascular anatomy, total concentration and oxygen saturation of hemoglobin (CHb and sO2), and blood flow in awake mice. From these hemodynamic measurements, two important metabolic parameters, oxygen extraction fraction (OEF) and the cerebral metabolic rate of oxygen (CMRO2), can be derived. Side-by-side comparison of the mouse brain under wakefulness and anesthesia revealed multifaceted cerebral responses to isoflurane, a volatile anesthetic widely used in preclinical research and clinical practice. Key observations include elevated cerebral blood flow (CBF) and reduced oxygen extraction and metabolism.

Stroke is the second leading cause of death worldwide. Rapid and precise diagnosis is essential to expedite clinical decision and improve functional outcomes in stroke patients; therefore, real-time imaging plays an important role to provide crucial information for post-stroke recovery analysis. In this study, based on the multi-wavelength laser and 18.5 MHz array-based ultrasound platform, a real-time handheld photoacoustic (PA) system was developed to evaluate cerebrovascular functions pre- and post-stroke in rats. Using this system, hemodynamic information such as cerebral blood volume (CBV) can be acquired for assessment. One rat stroke model (i.e., photothrombotic ischemia (PTI)) was employed for evaluating the effect of local ischemia. For achieving better intrinsic PA contrast, Vantage and COMSOL simulations were applied to optimize the light delivery (e.g., interval between two arms) from customized fiber bundle, while phantom experiment was conducted to evaluate the imaging performance of this system. Results of phantom experiment showed that hairs (~150 μm diameter) and pencil lead (500 μm diameter) can be imaged clearly. On the other hand, results of in vivo experiments also demonstrated that stroke symptoms can be observed in PTI model poststroke. In the near future, with the help of PA specific contrast agent, the system would be able to achieve blood-brain barrier leakage imaging post-stroke. Overall, the real-time handheld PA system holds great potential in disease models involving impairments in cerebrovascular functions.

Currently, the most researched noninvasive approach for monitoring neuro-electrical activity involves opticalfluorescence imaging, which suffers from limited imaging penetration. We propose an alternative approach, photoacoustic imaging (PAI) of biopotentials, that relies on transient absorption of light by voltage-sensitive probes and subsequent generation/detection of ultrasound. PAI-based voltage imaging approach can offer the same advantages as the fluorescence imaging in terms of sensitivity and molecular specificity, but it also can significantly extend the imaging depth. In this pilot study we are investigating the feasibility of photoacoustically visualizing biopotentials in rat pheochromocytoma (PC12) cells tagged with voltage-sensitive styrylpyridinium dye, RH795. A change in the intramembrane potential was induced in PC12 cells by adding tetraphenylborate (TPB) to the cell culture. A custommade absorption spectrophotometer was used to verify the change in optical absorption of RH795 dye as a result of TPBinduced electrical fields. Absorption spectra recorded before and after the addition of 100 μM TPB exhibited a wavelength shift of the absorption peak (approximately 510 nm to 550 nm) as well as an increase in the overall magnitude of absorption in the wavelength range of 500-1000 nm. The absorption spectral measurements indicated that RH795 is a good candidate as a voltage-sensitive dye for photoacoustically tracking changes in cell-membrane potential.

Functional magnetic resonance imaging (fMRI) has transformed our understanding of the brain’s functional organization. However, mapping subunits of a functional network using hemoglobin alone presents several disadvantages. Evoked and spontaneous hemodynamic fluctuations reflect ensemble activity from several populations of neurons making it difficult to discern excitatory vs inhibitory network activity. Still, blood-based methods of brain mapping remain powerful because hemoglobin provides endogenous contrast in all mammalian brains. To add greater specificity to hemoglobin assays, we integrated optical intrinsic signal(OIS) imaging with optogenetic stimulation to create an Opto-OIS mapping tool that combines the cell-specificity of optogenetics with label-free, hemoglobin imaging. Before mapping, titrated photostimuli determined which stimulus parameters elicited linear hemodynamic responses in the cortex. Optimized stimuli were then scanned over the left hemisphere to create a set of optogenetically-defined effective connectivity (Opto-EC) maps. For many sites investigated, Opto-EC maps exhibited higher spatial specificity than those determined using spontaneous hemodynamic fluctuations. For example, resting-state functional connectivity (RS-FC) patterns exhibited widespread ipsilateral connectivity while Opto-EC maps contained distinct short- and long-range constellations of ipsilateral connectivity. Further, RS-FC maps were usually symmetric about midline while Opto-EC maps displayed more heterogeneous contralateral homotopic connectivity. Both Opto-EC and RS-FC patterns were compared to mouse connectivity data from the Allen Institute. Unlike RS-FC maps, Thy1-based maps collected in awake, behaving mice closely recapitulated the connectivity structure derived using ex vivo anatomical tracer methods. Opto-OIS mapping could be a powerful tool for understanding cellular and molecular contributions to network dynamics and processing in the mouse brain.

Different brain regions exhibit complex information processing even at rest. Therefore, assessing temporal correlations between regions permits task-free visualization of their ‘resting state connectivity’. Although functional MRI (fMRI) is widely used for mapping resting state connectivity in the human brain, it is not well suited for ‘microvascular scale’ imaging in rodents because of its limited spatial resolution. Moreover, co-registered cerebral blood flow (CBF) and total hemoglobin (HbT) data are often unavailable in conventional fMRI experiments. Therefore, we built a customized system that combines laser speckle contrast imaging (LSCI), intrinsic optical signal (IOS) imaging and fluorescence imaging (FI) to generate multi-contrast functional connectivity maps at a spatial resolution of 10 μm. This system comprised of three illumination sources: a 632 nm HeNe laser (for LSCI), a 570 nm ± 5 nm filtered white light source (for IOS), and a 473 nm blue laser (for FI), as well as a sensitive CCD camera operating at 10 frames per second for image acquisition. The acquired data enabled visualization of changes in resting state neurophysiology at microvascular spatial scales. Moreover, concurrent mapping of CBF and HbT-based temporal correlations enabled in vivo mapping of how resting brain regions were linked in terms of their hemodynamics. Additionally, we complemented this approach by exploiting the transit times of a fluorescent tracer (Dextran-FITC) to distinguish arterial from venous perfusion. Overall, we demonstrated the feasibility of wide area mapping of resting state connectivity at microvascular resolution and created a new toolbox for interrogating neurovascular function.

In the last decade, Laser Speckle Contrast Imaging (LSCI) has been proposed and validated for imaging cerebral blood flow at the rodent brain surface in vivo. The technique relies on the calculation of the spatial speckle contrast, which is related to the velocity of scatterers (red blood cells). The implementation of the technique requires a partial craniotomy so that the brain tissues of interest can be illuminated with a laser diode. However, the studies of changes in the microcirculation during disease progression or treatment require longitudinal studies (i.e. imaging is done repeatedly over weeks or even months). Practically, the less invasive way to obtain such data is to image through the thinned skull without a craniotomy. However the presence of static scatterers (skull) will affect the speckle calculation and produce a bias in the estimation of the microcirculation changes. An extension to LSCI, termed Multi-Exposure Speckle Imaging (MESI) was proposed and validated a few years ago that address these limitations. It relies on a model of the speckle contrast as a function of the exposure time and the proportion of static scatterers. Here, we used MESI with the aim of repeatedly imaging the olfactory bulb of mice models of obesity. First, we have developed a MESI set up which was characterized on microfluidic flow phantoms with different flow-rates and channel diameters to simulate blood flow in animal model characteristics. Second, we show that MESI can discriminate flows in the presence of static scatterers and it can measure flow changes consistently. Finally we provide an in vivo validation of the technique in mice with and without a craniotomy.

Accurate and efficient reconstruction of hemodynamic changes is an important step towards the implementation of NIRS as an enhanced clinical tool for understanding oxygenation changes at various depths within the brain. Depth information could provide insight on how oxygen transported to the tissue. For this work, we ran Monte Carlo simulations to develop sensitivity profiles for various source-detector separations. The source-detector separations were based on our custom built 108 channel NIRS probe and consisted of separations of 15 mm, 30 mm, 36 mm, and 45 mm. We used the mesh-based Monte Carlo program MMCLAB (Fang et al. 2010) to acquire the sensitivity profiles. The sensitivity profiles consisted of a tetrahedral mesh which was converted to a regular grid in three-dimensional space. Then, the structural tensor was calculated for each voxel and the Hamilton-Jacobi equation was solved anisotropically for the tensor volume. As the result, the distance map was in same space as the calculated tensor volume. Using this distance map, we modeled the probabilistic path of photons. We then weighted the hemodynamic changes acquired by our NIRS probe according to the probabilistic path to reconstruct hemodynamic changes in the prefrontal area of the brain.

As with other imaging modalities, motion induces artifacts that can significantly corrupt optical neuroimaging data. While multiple methods have been developed for motion detection in individual NIRS measurement channels, the large measurement numbers present in multichannel fNIRS or high-density diffuse optical tomography (HD-DOT) systems, create an opportunity for detection methods that integrate over the entire field of view. Here, we leverage the inherent covariance among multiple NIRS measurements after pre-processing, to quantify motion artifacts by calculating the global variance in the temporal derivative (GV-TD) across all measurements (e.g. from the temporal derivative of each time-course, the method calculates root mean square across all measurements for each time point). This calculation is fast, automated, and identifies motion by incorporating global aspects of data instead of individual channels. To test the performance, we designed an experimental paradigm that intermixed controlled epochs of motion artifact with relatively motion-free epochs during a block design hearing-words language paradigm using a previously described HD-DOT system. We categorized 348 blocks by sorting the blocks based on the maximum of their GV-TD time-courses. Our results show that with a modest thresholding of the data, wherein we keep data with 0.66 of the full data set average GV-TD, we obtain a ~50% increase in the signal-to-noise. With noisier data, we expect the performance gains to increase. Further, the impact on resting state functional connectivity may also be more significant. In summary, a censoring threshold based on the GV-TD metric provides a fast and direct way for identifying motion artifacts.

Diffuse correlation spectroscopy (DCS) is being employed alongside near-infrared spectroscopy (NIRS) measurements to track the cerebral oxygen metabolic rate (CMRO2). However, both techniques employ diffusely reflected light that has traveled mostly through extracerebral tissues. Recent studies indicate that depth sensitivity profiles are different for NIRS vs DCS measurements, with DCS appearing to be more sensitive to the brain than NIRS methods for a given source-detector separation. This mismatch can lead to erroneous conclusions with respect to the amount and perhaps even the direction of change in CMRO2. Recently, our group and others have demonstrated the use of Monte Carlo (MC) based multi-layer, multi-distance fitting, which offers increased accuracy for complex tissue structures such as the adult brain. In this paper we employ a Monte Carlo light transport model based on a realistic head geometry that can be derived from MRI scans (if available) or approximated from head shape measurements. We consider DCS and CW-NIRS measurements taken at two or more distances and analyze simulated data generated using a fully segmented adult brain MRI scan. Through simulations, we explore the improvements offered by our method vs. processing the same measurements with a semi-infinite diffusion model and estimate the impact of errors in geometry and optical properties on relative blood flow and CMRO2 changes.

Brain-computer interfaces (BCIs) measuring electrical activity via electroencephalogram (EEG) have evolved beyond clinical applications to become wireless consumer products. Typically marketed for meditation and neu- rotherapy, these devices are limited in scope and currently too obtrusive to be a ubiquitous wearable. Stemming from recent advancements made in hearing aid technology, wearables have been shrinking to the point that the necessary sensors, circuitry, and batteries can be fit into a small in-ear wearable device. In this work, an ear-EEG device is created with a novel system for artifact removal and signal interpretation. The small, compact, cost-effective, and discreet device is demonstrated against existing consumer electronics in this space for its signal quality, comfort, and usability. A custom mobile application is developed to process raw EEG from each device and display interpreted data to the user. Artifact removal and signal classification is accomplished via a combination of support matrix machines (SMMs) and soft thresholding of relevant statistical properties.

Functional near-infrared spectroscopy (fNIRS) is suitable for measuring brain functions during neurorehabilitation because of its portability and less motion restriction. However, it is not known whether neural reconstruction can be observed through changes in cerebral hemodynamics. In this study, we modified an fNIRS system for measuring the motor function of awake monkeys to study cerebral hemodynamics during neurorehabilitation. Computer simulation was performed to determine the optimal fNIRS source–detector interval for monkey motor cortex. Accurate digital phantoms were constructed based on anatomical magnetic resonance images. Light propagation based on the diffusion equation was numerically calculated using the finite element method. The source–detector pair was placed on the scalp above the primary motor cortex. Four different interval values (10, 15, 20, 25 mm) were examined. The results showed that the detected intensity decreased and the partial optical path length in gray matter increased with an increase in the source-detector interval. We found that 15 mm is the optimal interval for the fNIRS measurement of monkey motor cortex. The preliminary measurement was performed on a healthy female macaque monkey using fNIRS equipment and custom-made optodes and optode holder. The optodes were attached above bilateral primary motor cortices. Under the awaking condition, 10 to 20 trials of alternated single-sided hand movements for several seconds with intervals of 10 to 30 s were performed. Increases and decreases in oxy- and deoxyhemoglobin concentration were observed in a localized area in the hemisphere contralateral to the moved forelimb.

The difference in signal-to-noise ratio (SNR) within functional near-infrared spectroscopic (fNIRS)-measurement channels makes it difficult to compare the significance of the signal amplitude in an individual channel against the baseline or against signals in other channels. The difference in SNR mainly originates from the difference in light loss due to the hair coverage or the optode–scalp contact. During the initial calibration of most commercial fNIRS equipment, the detected signals in different channels are differently amplified such that the system effectively utilizes a dynamic range for measurement. While different amplification rates among the channels realize almost equal signal intensities, they also differently amplify the detection noises. This results in different apparent noises in the fNIRS signals between channels. In order to level the SNRs in all the measurement channels, the system needs to equalize the light intensities received by the detectors instead of changing the signal amplification rates. To realize this novel procedure for leveling the SNR among the channels, we developed an fNIRS system equipped with an optical attenuator at each source and detector. A systematic procedure for modulating the attenuators to level SNR over all channels was mathematically formulated, and the procedure was examined using an optical phantom with a surface covered with air.

pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical reactivation imaging in conjunction with EGFP or EYFP.

Resonance Raman (RR) spectroscopy offers a novel Optical Biopsy method in cancer discrimination by a means of enhancement in Raman scattering. It is widely acknowledged that the RR spectrum of tissue is a superposition of spectra of various key building block molecules. In this study, the Resonance Raman (RR) spectra of human metastasis of lung cancerous and normal brain tissues excited by a visible selected wavelength at 532 nm are used to explore spectral changes caused by the tumor evolution. The potential application of RR spectra human brain metastasis of lung cancer was investigated by Blind Source Separation such as Principal Component Analysis (PCA). PCA is a statistical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components (PCs). The results show significant RR spectra difference between human metastasis of lung cancerous and normal brain tissues analyzed by PCA. To evaluate the efficacy of for cancer detection, a linear discriminant analysis (LDA) classifier is utilized to calculate the sensitivity, and specificity and the receiver operating characteristic (ROC) curves are used to evaluate the performance of this criterion. Excellent sensitivity of 0.97, specificity (close to 1.00) and the Area Under ROC Curve (AUC) of 0.99 values are achieved under best optimal circumstance. This research demonstrates that RR spectroscopy is effective for detecting changes of tissues due to the development of brain metastasis of lung cancer. RR spectroscopy analyzed by blind source separation may have potential to be a new armamentarium.