This PDF file contains the front matter associated with SPIE Proceedings Volume 10481, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Cerebral autoregulation (CA) is a homeostatic mechanism that maintains a relatively constant cerebral blood flow (CBF) in the presence of changes in the cerebral perfusion pressure (CCP), defined as the difference between mean arterial pressure (MAP) and intracranial pressure (ICP). Given the importance of adequate and consistent brain perfusion, CA is critical for brain viability and is known to be impaired in a number of neurological disorders. Global brain measurements of dynamic CA have been performed with transcranial Doppler ultrasound (to sense the blood flow velocity in the middle cerebral artery) and finger plethysmography (to measure systemic MAP as a surrogate for CCP). Optical methods offer the advantage of providing local measurements of cerebral blood flow and CA, thus allowing for local assessment and spatial mapping of CA. Optical techniques for the non-invasive assessment of CA include near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS). I will describe our approach to CA assessment with NIRS, complemented by the novel technique of coherent hemodynamics spectroscopy (CHS), and our findings of the expected enhancement in CA during hyperventilation-induced hypocapnia. I will also report dynamic traces of local CBF measured with NIRS-CHS and DCS during transient changes in MAP. Optical techniques offer the potential to address the challenge of continuous monitoring of local cerebral autoregulation at the bedside and in a critical care environment.

Cortical capillary blood flow and oxygenation are highly heterogeneous. Mapping absolute capillary blood flow and oxygenation along capillary path is a key step towards understanding how oxygen is transported and delivered in a complex microvascular network to enable adequate tissue oxygenation. In this work, we applied two-photon microscopic imaging of intravascular oxygen partial pressure (PO2) to measure both oxygen concentration and red blood cell (RBC) flux in cortical arterioles, capillaries, and venules. Imaging was performed in awake, head-restrained C57BL/6 mice (n=15), through a chronic sealed cranial window centered over the E1 whisker barrel. We obtained a detailed mapping of the resting state cortical microvascular PO2 in all arterioles and venules, and both PO2 and RBC flux in most capillaries down to 600 μm depth from the cortical surface (n=6,544 capillaries across all mice). Capillary RBC speed and density were also extracted and all measurements were co-registered with the microvascular angiograms. We characterized the distributions of capillary PO2 and flow as a function of branching order and cortical depth. The results show strong positive correlation between oxygenation and flow in the capillary segments, with an increased correlation in downstream capillaries. We have also observed homogenization of both oxygenation and flow in deeper cortical layers, which may imply a mechanism to improve oxygen delivery without increasing global blood flow in the area with increased metabolism.

Amyloid depositions in the brain represent the characteristic hallmarks of Alzheimer’s disease (AD) pathology. The abnormal accumulation of extracellular amyloid-beta (Aβ) and resulting toxic amyloid plaques are considered to be responsible for the clinical deficits including cognitive decline and memory loss. In vivo two-photon fluorescence imaging of amyloid plaques in live AD mouse model through a chronic imaging window (thinned skull or craniotomy) provides a mean to greatly facilitate the study of the pathological mechanism of AD owing to its high spatial resolution and long-term continuous monitoring. However, the imaging depth for amyloid plaques is largely limited to upper cortical layers due to the short-wavelength fluorescence emission of commonly used amyloid probes. In this work, we reported that CRANAD-3, a near-infrared (NIR) probe for amyloid species with excitation wavelength at 900 nm and emission wavelength around 650 nm, has great advantages over conventionally used probes and is well suited for twophoton deep imaging of amyloid plaques in AD mouse brain. Compared with a commonly used MeO-X04 probe, the imaging depth of CRANAD-3 is largely extended for open skull cranial window. Furthermore, by using two-photon excited fluorescence spectroscopic imaging, we characterized the intrinsic fluorescence of the “aging pigment” lipofuscin in vivo, which has distinct spectra from CRANAD-3 labeled plaques. This study reveals the unique potential of NIR probes for in vivo, high-resolution and deep imaging of brain amyloid in Alzheimer’s disease.

High-resolution optical longitudinal cortical imaging usually uses cranial window, which involves removing a skull portion and sealing the exposed brain area with a transparent cover glass, allowing ballistic photons to reach the cortex with minimal disturbance of the brain function. It enables obtaining high-resolution brain images in extended periods of time for long-term neuronal activity studies using confocal and two-photon microscopies. Photoacoustic microscopy (PAM), as the only imaging method that directly measure absorption contrast, is a complementary functional imaging method to provide absorption related brain information, such as total concentration of hemoglobin and oxygen saturation of hemoglobin. However, the use of traditional piezoelectric transducers (PZT) to collect ultrasound signal greatly limits the versatility of PAM. Though highly sensitive, PZT transducers are usually bulky and optically opaque. It blocks the light and is hard to be inserted into the limited distance between the optical objective and imaging sample, which are normally less than one millimeter when using a high- numerical aperture (NA) objective to achieve submicron resolution. Here, we developed a simple and cost-efficient soft nanoimprint lithography (NIL) process to fabricate fully embedded micro-ring resonator ultrasound detectors on optically transparent substrates, and integrated the detector onto a cranial window, making cranial window itself an ultrasonic detector. We implanted this functional cranial window on mouse head and achieved longitudinal monitoring of cortex vasculature using PAM. Our low-cost, disposable, and optically transparent detector may potentially reshape the longitudinal functional brain imaging using PAM in small animals.

Quantitative and scalable whole-brain neuroanatomical mapping, with cellular resolution and molecular specificity, poses significant technological challenges. Indeed, a high image quality must be preserved reliably across the entire specimen and not only in a few representative volumes. On the other hand, robust and automated image analysis methods must be appropriately scalable to teravoxel datasets. Here, we present an experimental pipeline, involving tissue clearing, high-resolution light-sheet microscopy, volume registration to atlas, and deep learning strategies for image analysis, allowing the reconstruction of 3D maps of selected cell types in the whole mouse brain. We employed RAPID autofocusing [Silvestri et al., submitted] to keep the system sharply in focus throughout the entire mouse brain, without reducing the microscope throughput. Images were spatially anchored to reference atlas using semi-automatic tools (xNII family, http://www.nesys.uio.no). Finally, we used novel high-throughput tools for image processing, including deep learning strategies [Frasconi et al., 2014] to localize single neurons with high accuracy. By applying our pipeline to transgenically-labeled samples, we can produce an atlas of spatial distribution of genetically-defined cell types. Besides being a valuable reference for neurobiologists, these datasets can be used to build realistic simulations of neuronal functioning, such as in the Human Brain Project.

High resolution imaging of whole rodent brains using serial OCT scanners is a promising method to investigate microstructural changes in tissue related to the evolution of neuropathologies. Although micron to sub-micron sampling resolution can be obtained by using high numerical aperture objectives and dynamic focusing, such an imaging system is not adapted to whole brain imaging. This is due to the large amount of data it generates and the significant computational resources required for reconstructing such volumes. To address this limitation, a dual resolution serial OCT scanner was developed. The optical setup consists in a swept-source OCT made of two sample and reference arms, each arm being coupled with different microscope objectives (3X / 40X). Motorized flip mirrors were used to switch between each OCT arm, thus allowing low and high resolution acquisitions within the same sample. The low resolution OCT volumes acquired with the 3X arm were stitched together, providing a 3D map of the whole mouse brain. This brain can be registered to an OCT brain template to enable neurological structures localization. The high resolution volumes acquired with the 40X arm were also stitched together to create local high resolution 3D maps of the tissue microstructure. The 40X data can be acquired at any arbitrary location in the sample, thus limiting storage-heavy high resolution data to application restricted to specific regions of interest. By providing dual-resolution OCT data, this setup can be used to validate diffusion MRI with tissue microstructure derived metrics measured at any location in ex vivo brains.

Drosophila is an important model animal to study connectomics since its brain is complicated and small enough to be mapped by optical microscopy with single-cell resolution. Compared to other model animals, its genetic toolbox is more sophisticated, and a connectome map with single-cell resolution has been established, serving as an invaluable reference for functional connectome study. Two-photon microscopy (2PM) is now the most popular tool to study functional connectome by taking the advantages of low photobleaching, subcellular resolution and deep penetration depth. However, using GFP-labeling with excitation wavelength ~ 920-nm, the reported penetration depths in a living Drosophila brain are limited to ~ 100-μm, which are much smaller than that in living mouse or zebrafish brains. The underlying reason is air vessels, i.e., trachea, instead of blood vessels, are responsible for oxygen exchange in Drosophila brains. The trachea structures induce extraordinarily strong scattering and aberration since the air/tissue refractive index difference is much larger than blood/tissue. By expelling the air inside trachea, whole Drosophila brain can be penetrated by 2PM without difficulty. However, the Drosophila is not alive anymore. Here, three-photon microscopy based on a 1300-nm laser is demonstrated to penetrate a living Drosophila brain with single-cell resolution. The long wavelength intrinsically reduces scattering, when combined with normal dispersion of brain tissue, aberration from trachea/tissue interface is reduced to some extent. As a result, the penetration depth is improved more than twice using 1300-nm excitation. This technique is believed to significantly contribute on functional connectome studies in the future.

Photoacoustic tomography (PAT), combining optical and ultrasonic waves via the photoacoustic effect, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution imaging across the length scales of organelles, cells, tissues, and organs with consistent contrast. PAT has the potential to empower multiscale biology research and accelerate translation from microscopic laboratory discoveries to macroscopic clinical practice. PAT can image the entire brain of a rat or mouse with optical contrast in vivo. Broad applications include imaging of the breast, brain, skin, esophagus, colon, vascular system, and lymphatic system in both humans and animals.

Neuronal activity occurs simultaneously and in a highly coordinated fashion in many different areas across the brain. Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with the existing neuroimaging methods that are limited in their ability to image large tissue volumes at high speeds. Genetically encoded calcium indicators (GECIs) that modulate their fluorescence intensity as a function of intracellular calcium concentrations are powerful tools for the observation of large neuronal networks. Optoacoustic imaging has been shown capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep in mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as lack of activity labels excitable in the near-infrared window. We developed and validated an isolated whole mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions and exhibit functional activity for several hours to several days. An optoacoustic imaging system coupled to a superfusion system was further devised and used for rapid volumetric monitoring of calcium dynamics in the brain evoked using an epilepsy-inducing drug. The new technique captures calcium fluxes as true 3D information across the entire brain with temporal resolution of 10ms and spatial resolution of 150µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. The system could be readily adapted to work with future generations of far-red- and near-infrared GECIs.

Optical visualization of Alzheimer’s disease (AD) pathological changes is crucial to facilitate exploration of disease mechanism and treatment. We developed cryo-micro-optical sectioning tomography (cryo-MOST) to acquire brainwide map of senile plaques. Using intrinsic fluorescence emission intensified under ultra-low temperature, we accomplished senile plaque visualization at a micron-level resolution. A whole-brain coronal distribution of senile plaque in a transgenic mouse was successfully acquired without any exogenous dye. We believe cryo-MOST would be a potential tool for understanding neurodegenerative disease mechanism and evaluating drug efficacy.

The brain-wide reconstruction of neuronal population is an indispensible step towards exploring the complete structure of neuronal circuits, a central task that underlies the structure-function relation in neuroscience. Recent advances in molecular labeling and imaging techniques enable us to collect the whole mouse brain imaging dataset at cellular resolution, including the morphological information of neurons across different brain region or even the whole brain. Reconstruction of these neurons poses substantial challenges, and at presents there is no tool for high-speed achieving this reconstruction close to human performance. Here, we presented a tool for filling in the blanks. The tool mainly contains the following function modules: 3D visualization of large-scale imaging dataset, automated reconstruction of neurons, manual editing of the reconstructions at local and global scale. In this tool, in the framework of our previous tools (NeuroGPS-Tree and SparseTracer), the two identifying models were constructed for boosting the automatic level of the reconstruction. One is used to identify the weak signals from inhomogeneous backgrounds and the other is used to identify closely packed neurites. This tool can be suitable for the different big-data formats and can make the dataset be fastly read into memory for the reconstruction. The manual editing module in this tool can correct the errors drawn from above automated algorithms. And thus helps to achieve the reconstruction closer to human performance. We demonstrated the features of our tool on various kinds of sparsely labelled datasets. The results indicated that without loss of the reconstruction accuracy, our tool has a 7-10 folds speed gain over the commercial software that provides the manual reconstruction.

Optical coherence tomography angiography (OCTA) is a promising imaging modality that enables an in vivo label-free, high-resolution and high-contrast visualization of three-dimensional biological microvasculature. The blood flow contrast in OCTA is achieved by mathematically distinguishing the dynamic flow from the static surrounding tissue. However, the residual surrounding tissue remains as the background in the angiogram, which severely hinders the interpretation and quantification of the angiographic outcomes. The current temporal, wavelength, angular and spatial averaging approaches have been employed to enhance the flow contrast by trading imaging time and resolution for multiple independent measurements. Our study has further demonstrated that these averaging approaches are equivalent in principle, offering almost the same flow contrast improvement as the number of averages increases. Given a sufficient number, an ideal flow contrast can be achieved, while the cost of imaging time or resolution is unaffordable for any individual averaging approach alone. Thus, we have proposed a hybrid averaging strategy for a desired flow contrast by cost apportionment. It is demonstrated that, compared with any individual approach, hybrid averaging is able to offer a desired flow contrast without severe degradation of imaging time and resolution. In addition, making use of the extended range of a VCSEL based swept source OCT, an angular averaging approach by path length encoding is also demonstrated for flow contrast enhancement. This study is beneficial to providing useful guidance for the design of OCTA and facilitating the interpretation of OCT angiograms in clinical applications.

Multicore fiber bundles for imaging and stimulating optogenetically modified neurons have been largely adopted in neurophotonics research. They allow for directed, single-cell stimulation and imaging of neuronal activity. An inherent limitation of these bundles is the presence and detection of the empty space between individual fibers, resulting in a loss of significant amounts of data, and reduced image quality due to pixilation effects. We propose a novel approach and algorithm to depixelation and image reconstruction from fiber bundles that utilizes multiple image frames collected during on-axis fiber bundle rotation. The approach involves first acquiring the Fourier transform of a stationary, unrotated image, followed by its rotated counterparts. The phase information from each image is then acquired, cross-correlated, and the angle of rotation determined from this correlation. Rotated images are then weighed and summed to generate a final reconstructed, depixelated image. Simulations were initially performed using Matlab demo images. Experimentation was done with a resolution chart, and thereafter with a cell culture. 488 nm and 561 nm continuous wave laser sources (Coherent, Inc.) were used for imaging GCaMP6s and C1V1-mCherry, respectively, in hippocampal neuronal cultures. The light sources were coupled to a multicore fiber bundle (Schott, 1534702) containing 4,200, 7.5 µm fibers. Cell cultures were prepared from 2 day old transgenic mice (GCaMP6s, Jackson Labs) transfected with C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford). The results demonstrate this as an effective technique alongside fiber bundle imaging, serving as a useful and powerful tool for removing undesired artifacts associated with these fibers.

Precision sensing needs to overcome a gap of a single atomic step height standard. In response to the cutting-edge challenge, a heterosingle molecular nanomedicine crystal was developed wherein a nanomedicine crystal height less than 1 nm was designed and selfassembled on a substrate of either a highly ordered and freshly separated graphite or a N-doped silicon with hydrogen bonding by a home-made hybrid system of interacting single bioelectron donor-acceptor and a single biophoton donor-acceptor according to orthogonal mathematical optimization scheme, and an atomic spatial resolution conducting atomic force microscopy (C-AFM) with MHz signal processing by a special transformation of an atomic force microscopy (AFM) and a scanning tunneling microscopy (STM) were employed, wherein a z axis direction UV-VIS laser interferometer and a feedback circuit were used to achieve the minimized uncertainty of a micro-regional structure height and its corresponding local differential conductance quantization (spin state) process was repeatedly measured with a highly time resolution, as well as a pulsed UV-VIS laser micro-photoluminescence (PL) spectrum with a single photon resolution was set up by traceable quantum sensing and metrology relied up a quantum electrical triangle principle. The coupling of a single bioelectron conducting, a single biophoton photoluminescence, a frequency domain temporal spin phase in nanomedicine crystal-inspired sensing methods and sensor technologies were revealed by a combination of C-AFM and PL measurement data-based mathematic analyses^1-3, as depicted in Figure 1 and repeated in nanomedicine crystals with a single atomic height. It is concluded that height-current-phase uncertainty correlation pave a way to develop a brain imaging and a single atomic height standard, quantum sensing, national security, worldwide impact^1-3 technology and beyond.

Super-resolution localization microscopy (SRLM), including PALM, STORM, dSTORM and many others, achieves ultra-high spatial resolution up to 20~30 nanometers by positioning and reconstructing single molecules from thousands or even tens of thousands of raw images. As intrinsically a wide-field imaging technique, SRLM has the advantage of increasing field-of-view (FOV) without sacrificing either imaging speed or spatial resolution. Currently, limited by the number of active pixels in EMCCD cameras (typically 512 x 512), the maximum FOV of popular SRLM is approximately 50 um x 50 um at the sample plane. Such an FOV is insufficient for observing many biological phenomena which are best interpreted in large FOV, for example, volumetric mapping of synaptic connectivity at multiple scales. In this talk, we will report our recent progresses in the technology development and applications of SRLM with large FOV. We will firstly report the imaging performance of a back-illuminated sCMOS cameras with 95% QE for SRLM. Then, we discuss a high-power homogeneous illumination system which is capable of providing sufficient illumination intensity and excellent illumination homogeneity for SRLM with large FOV. Finally, we present some preliminary results of using large FOV SRLM in mapping synaptic connectivity at multiple scales.

Optical clearing methods are highly in demand in organism-level biomedical system research since they can facilitate deep optical imaging by reducing light scattering in tissue and then enable three-dimensional signal visualization and quantification of tissues. While the previously reported optical clearing methods have addressed some of six key issues (i.e. transparency, efficiency, reproducibility, preservation of emission from fluorescence proteins, preservation of membrane integrity, and the ease of operation), none has yet addressed all of them. Here, we present a new, convenient, inexpensive and reproducible approach to optical clearing, termed UbasM, providing unprecedented performance in terms of clearing rate, the ease of operation and satisfactory fluorescence protein/membrane integrity preservation while achieving sufficient transparency to permit 3D volumetric imaging.

Interferometric near-infrared spectroscopy (iNIRS) is a recently introduced time-of-flight- (TOF-) resolved sensing method for quantifying optical and dynamical properties of turbid media non-invasively. iNIRS measures the interference spectrum of light traversing a turbid medium using a rapidly tunable, or frequency swept, light source. While the modality was successfully demonstrated in vivo in the nude mouse brain for monitoring absorption, reduced scattering, and blood flow index, translation towards human measurements requires improving light collection efficiency. Particularly, interrogating cortical tissue beneath the adult human scalp and skull remains challenging due to the limited core size and throughput of the single mode fiber currently used for detection. To tackle this problem, we implement a short to null source-detector separation geometry setup to significantly improve the number of detected diffuse photons. We discuss both hardware and post-processing improvements to isolate the desired diffuse signal from the large, non-diffuse and specular signals. Furthermore, key improvements in the iNIRS optical system, including higher TOF resolution (22-60 ps), optimized dynamic range (36-47 dB), faster sweep rate (50-500 kHz), and a technique for combining the forward and backward sweeps to double the effective optical field autocorrelation sampling rate, are presented. These allow for more precise and quantitative extraction of in vivo optical properties and TOF-resolved dynamics at long path lengths. Collectively, these key advances in the technology pave the way for translating iNIRS towards non-invasive, real-time, and quantitative measurements of oxygen metabolism and blood perfusion in deep human tissues.

It has been observed that there is a low-frequency oscillation (LFO) around 0.1 Hz in cerebral hemodynamics related to brain activity. Since functional near-infrared spectroscopy (fNIRS) is a novel technique to monitor hemodynamic responses noninvasively, we applied it to detect LFOs of cerebral hemodynamic parameters, such as oxyhemoglobin and deoxyhemoglobin, during prolonged driving. We performed an experiment lasting for 7 hours and an experimental test was done every hour and 8 times altogether. 7 subjects were recruited and the data of 3 of them were analyzed. By means of Fourier transformation, the amplitude of the three parameters during each test at 0.1 Hz in frequency domain was extracted. The results showed an increasing trend in the 0.1 Hz amplitudes of the three hemodynamic parameters during 7 hours' simulated driving test. Our findings indicated the potential of LFOs of prefrontal cerebral hemodynamics in brain research and brain function evaluation.

Cardiac arrest (CA) affects over 500,000 people in the United States. Although resuscitation efforts have improved, poor neurological outcome is the leading cause of morbidity in CA survivors, and only 8.3% of out-of-hospital CA survivors have good neurological recovery. Therefore, a detailed understanding of the brain before, during, and after CA and resuscitation is critical. To provide a more complete picture of CBF dynamics associated with CA and resuscitation, we postulate that both temporal and spatial CBF dynamics must be understood. To investigate spatiotemporal dynamics, we used laser speckle imaging (LSI) to image rats that underwent either 5- or 7-min asphyxial CA, followed by cardiopulmonary resuscitation until return of spontaneous circulation (ROSC). During induction of global cerebral ischemia through CA, we observed two time periods during which a decrease in CBF propagates in space in a cranial window over the right hemisphere. The first time-period is during CA and the second after the hyperemic peak, but before CBF plateaus at a hypoperfused state post-ROSC. During CA, the decrease in CBF propagates from the lateral region of the brain to the medial region of the brain. Conversely, post-ROSC, the decrease in CBF propagates from the medial region of the brain to the lateral region of the brain. We postulate that study of spatiotemporal dynamics in a global cerebral ischemia model may lead to important insight into our understanding of cerebral function during and after resuscitation from CA, which may provide clinicians with knowledge that can lead to improvements in neurological outcome.

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Peripheral nerves connect and relay information between the central nervous system and its target organs. Small arteries traverse the epineurium and are responsible for supplying blood to the axons and cells within the nerves. Constriction or damage to these vessels can reduce perfusion leading to ischemic insults. Peripheral nerve electrostimulation has been approved for the treatment of epilepsy, depression and migraines, and is also being studied for the treatment of rheumatoid arthritis, Crohn’s disease, polycystic ovary syndrome, and type II diabetes. While the safety and efficacy of currently approved medical devices is well established, next generation devices may require novel stimulation parameters that pose additional risks. Therefore it is important to develop new methods to assess stimulation-induced nerve injury. To that end, we have begun to explore optical imaging based biomarkers, including optical coherence tomography angiography (OCT-A) to quantify changes in vascular morphology and blood flow during stimulation. We imaged the rat sciatic nerve in vivo with a 1300 nm OCT-A system. A 3-D printed nerve stabilizer with embedded platinum disc electrode was used to align the nerve for imaging during electrostimulation. Electrostimulation at either 40 or 400 µC/cm2 was applied for 1 hour. Images were acquired before, during and after stimulation. With higher electrostimulation parameters, blood vessels close to electrode site showed constriction. Immunohistochemical assessment was performed to correlate nerve injury to observed vascular changes. Optical imaging biomarkers have the potential to help assess the safety of novel electrodes and electrostimulation paradigms.

Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. Post-mortem studies showed neurodegeneration involving white matter components in the cerebral lobes, the cerebellar peduncles and the more distal cranial nerves in human patients. However, the progression of SCA1 in the brain remains unclear. We present the study of white matter atrophy of SCA1 mouse models using serial optical coherence scanner (SOCS). SOCS consists of a polarization sensitive optical coherence tomography and a tissue slicer (vibratome) with associated controls for serial imaging. The optical system has 5.5 µm axial resolution and utilizes a scan lens or a water-immersion microscope objective to provide 10 µm or 4 µm lateral resolution, respectively. Brain imaging with SOCS showed that the reflectivity contrast portrays morphology, and the polarization contrasts primarily probe myelinated nerve fibers in the white matter. In the cerebellum, the cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. We use SOCS to image the cerebellums of SCA1 mouse models. Data have been acquired from multiple sections at different age groups. The label-free contrasts show the pathological changes in molecular layer in SCA1 mouse models. White matter size in midline section was quantified at different time points to show white matter degeneration. Moment analysis for retardance contrast and distribution of axis orientation contrast reveal white matter atrophy. High-resolution (4 µm) SOCS visualizes the atrophy of fine features in midline sagittal cerebellum sections as well.

Playing a dance video game (DVG) requires fine temporal control of foot positions based on simultaneous visuoauditory integration. Despite the highly-demanding nature of its cognitive processes, DVG could offer promising exercise opportunities for elderly people to maintain their cognitive abilities due to its strong adherence. Using functional near-infrared spectroscopy, we have previously shown that DVG play with the foot activates prefrontal and temporoparietal cortices. However, it is still in debate whether this brain-stimulatory effect of DVG could also be maintained in case that DVG is played with the hand by people who have difficulty to play DVG in a standing position. We therefore investigated the regional brain activity of 12 healthy, right-handed young-adults when they played DVG with their dominant hand and foot. We found that the DVG-related hemodynamic activity was comparable in the prefrontal area regardless of the appendages while that was significantly smaller in case of playing with the hand related to the foot in the left superior/middle temporal gyrus (S/MTG). A similar trend was also observed in the right S/MTG. These results suggest that the motor preparatory function mediated by the prefrontal cortices is equally employed regardless of appendages while more cognitive load is required in the temporal cortices with foot-played DVG, possibly to integrate visual, auditory, and proprioceptive information. Hand-played DVG may partially substitute foot-played DVG in the sense of cognitive training in the elderly.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by short-term memory loss and cognitive inabilities. This work seeks to study the effects of voluntary exercise on the change in oxygen delivery in awake mice models of Alzheimer’s disease by monitoring brain tissue oxygenation. Experiments were performed on Young (AD_Y, 3-4 months, n=8), Old (AD_O, 6-7 months, n=8), and Old with exercise (AD_OEX, 6-7 months, n=8) transgenic APPPS1 mice and their controls. Brain tissue oxygenation was measured by two photon phosphorescence lifetime microscopy on the left sensory motor cortex. We found that the average tissue PO2 decreased with age but were regulated by exercise. The results suggest a potential for exercise to improve brain function with age and AD.

Functional monitoring of highly-localized deep brain structures is of great interest. However, due to light scattering, optical methods have limited depth penetration or can only measure from a large volume. In this research, we demonstrate continuous measurement of hemodynamics in different cortical layers in response to thalamic deep brain stimulation (DBS) using a single fiber optical system. A 200-μm-core-diameter multimode fiber is used to deliver and collect light from tissue. The fiber probe can be stereotaxically implanted into the brain region of interest at any depth to measure the di use reflectance spectra from a tissue volume of 0.02-0.03 mm³ near the fiber tip. Oxygenation is then extracted from the reflectance spectra using an algorithm based on Monte Carlo simulations. Measurements were performed on the surface (cortical layer I) and at 1.5 mm depth (cortical layer VI) of the motor cortex in anesthetized rats with thalamic DBS. Preliminary results revealed the oxygenation changes in response to DBS. Moreover, the baseline as well as the stimulus-evoked change in oxygenation were different at the two depths of cortex.

Recently, a variety of tissue optical clearing techniques have been developed to reduce light scattering for imaging deeper and three-dimensional reconstruction of tissue structures. Combined with optical imaging techniques and diverse labeling methods, these clearing methods have significantly promoted the development of neuroscience. However, most of the protocols were proposed aiming for specific tissue type. Though there are some comparison results, the clearing methods covered are limited and the evaluation indices are lack of uniformity, which made it difficult to select a best-fit protocol for clearing in practical applications. Hence, it is necessary to systematically assess and compare these clearing methods. In this work, we evaluated the performance of seven typical clearing methods, including 3DISCO, uDISCO, SeeDB, ScaleS, Clear^T2, CUBIC and PACT, on mouse brain samples. First, we compared the clearing capability on both brain slices and whole-brains by observing brain transparency. Further, we evaluated the fluorescence preservation and the increase of imaging depth. The results showed that 3DISCO, uDISCO and PACT posed excellent clearing capability on mouse brains, ScaleS and SeeDB rendered moderate transparency, while Clear^T2 was the worst. Among those methods, ScaleS was the best on fluorescence preservation, and PACT achieved the highest increase of imaging depth. This study is expected to provide important reference for users in choosing most suitable brain optical clearing method.

Dynamic Light Scattering-Optical Coherence Tomography (DLS-OCT) takes the advantages of using DLS to measure particle flow and diffusion within an OCT resolution-constrained 3D volume, enabling the simultaneous measurements of absolute RBC velocity and diffusion coefficient with high spatial resolution. In this work, we applied DLS-OCT to measure both RBC velocity and the shear-induced diffusion coefficient within penetrating venules of the somatosensory cortex of anesthetized mice. Blood flow laminar profile measurements indicate a blunted laminar flow profile, and the degree of blunting decreases with increasing vessel diameter. The measured shear-induced diffusion coefficient was proportional to the flow shear rate with a magnitude of ~ 0.1 to 0.5 × 10^-6 mm² . These results provide important experimental support for the recent theoretical explanation for why DCS is dominantly sensitive to RBC diffusive motion.

Image segmentation plays an important role in multimodality imaging, especially in fusion structural images offered by CT, MRI with functional images collected by optical technologies or other novel imaging technologies. Plus, image segmentation also provides detailed structure description for quantitative visualization of treating light distribution in the human body when incorporated with 3D light transport simulation method. Here we used image enhancement, operators, and morphometry methods to extract the accurate contours of different tissues such as skull, cerebrospinal fluid (CSF), grey matter (GM) and white matter (WM) on 5 fMRI head image datasets. Then we utilized convolutional neural network to realize automatic segmentation of images in a deep learning way. We also introduced parallel computing. Such approaches greatly reduced the processing time compared to manual and semi-automatic segmentation and is of great importance in improving speed and accuracy as more and more samples being learned. Our results can be used as a criteria when diagnosing diseases such as cerebral atrophy, which is caused by pathological changes in gray matter or white matter. We demonstrated the great potential of such image processing and deep leaning combined automatic tissue image segmentation in personalized medicine, especially in monitoring, and treatments.

Light-sheet fluorescence microscopy (LSFM) uses an additional laser-sheet to illuminate selective planes of the sample, thereby enabling three-dimensional imaging at high spatial-temporal resolution. These advantages make LSFM a promising tool for high-quality brain visualization. However, even by the use of LSFM, the spatial resolution remains insufficient to resolve the neural structures across a mesoscale whole mouse brain in three dimensions. At the same time, the thick-tissue scattering prevents a clear observation from the deep of brain. Here we use multi-view LSFM strategy to solve this challenge, surpassing the resolution limit of standard light-sheet microscope under a large field-of-view (FOV). As demonstrated by the imaging of optically-cleared mouse brain labelled with thy1-GFP, we achieve a brain-wide, isotropic cellular resolution of ~3μm. Besides the resolution enhancement, multi-view braining imaging can also recover complete signals from deep tissue scattering and attenuation. The identification of long distance neural projections across encephalic regions can be identified and annotated as a result.