This PDF file contains the front matter associated with SPIE Proceedings Volume 8928, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

Optical coherence microscopy (OCM) provides real-time, in-vivo, three-dimensional, isotropic micron-resolution structural and functional characterization of tissue, cells, and other biological targets. Optical coherence angiography (OCA) also provides visualization and quantification of vascular flow via speckle-based or phase-resolved techniques. Performance assessment of neuroprosthetic systems, which allow direct thought control of limb prostheses, may be aided by OCA. In particular, there is a need to examine the underlying mechanisms of chronic functional degradation of implanted electrodes. Angiogenesis, capillary network remodeling, and changes in flow velocity are potential indicators of tissue changes that may be associated with waning electrode performance. The overall goal of this investigation is to quantify longitudinal changes in vascular morphology and capillary flow around neural electrodes chronically implanted in mice. We built a 1315-nm OCM system to image vessels in neocortical tissue in a cohort of mice. An optical window was implanted on the skull over the primary motor cortex above a penetrating shank-style microelectrode array. The mice were imaged bi-weekly to generate vascular maps of the region surrounding the implanted microelectrode array. Acute effects of window and electrode implantation included vessel dilation and profusion of vessels in the superficial layer of the cortex (0-200 μm). In deeper layers surrounding the electrode, no qualitative differences were seen in this early phase. These measurements establish a baseline vascular tissue response from the cortical window preparation and lay the ground work for future longitudinal studies to test the hypothesis that vascular changes will be associated with chronic electrode degradation.

Single band coherent anti-Stokes Raman scattering (CARS) microscopy within the CH-stretching region is applied to detect individual cells and nuclei of human brain tissue and brain tumors – an information which allows for histopathologic grading of the tissue. The CARS image contrast within the C-H-stretching region correlated to the tissue composition. Based on the specific application example of identifying nuclei within (coherent) Raman images of neurotissue sections, we shall derive general design parameters for lasers optimally suited to serve in a clinical environment and discuss the potential of recently developed methods to analyze spectrally resolved CARS images and image segmentation algorithms.

Gliomas are extremely infiltrative type of brain cancers, the borders of which are difficult to locate. Gliomas largely consist of tumors of astrocytic or oligodendroglial lineage. Usually stereotactic surgery is performed to obtain tumor tissue sample. Complete excision of these tumors with preservation of uninvolved normal areas is important during brain tumor surgeries. The present study was undertaken to explore feasibility of classifying abnormal and normal glioma tissues with Raman spectroscopy (RS). RS is a nondestructive vibrational spectroscopic technique, which provides information about molecular composition, molecular structures and molecular interactions in tissue. Postoperated 33 (20-abnormal and 13-normal) gliomas tissue samples of different grades were collected under clinical supervision. Five micron section from tissue sample was used for confirmatory histopathological diagnosis while the remaining tissue was placed on CaF2 window and spectra were acquired using a fiberoptic-probe-coupled HE-785 Raman-spectrometer. Spectral acquisition parameters were laser power-80mW, integration-20s and averaged over 3 accumulations. Spectra were pre-processed and subjected to unsupervised Principal-Component Analysis (PCA) to identify trends of classification. Supervised PC-LDA (Principal-Component-Linear-Discriminant Analysis) was used to develop standard-models using spectra of 12 normal and abnormal specimens each. Leave-one-out crossvalidation yielded classification-efficiency of 90% and 80% for normal and abnormal conditions, respectively. Evaluation with an independent-test data-set comprising of 135 spectra of 9 samples provided sensitivity of 100% and specificity of 70%. Findings of this preliminary study may pave way for objective tumor margin assessment during brain surgery.

Fluorescence-guidance is a useful adjunct to maximize brain tumor resection but current commercial systems are limited by subjective assessment of fluorescence, low sensitivity and non-spectrally-resolved detection. We present a quantitative, spectrally-resolved system integrated onto a commercial neurosurgical microscope that performs spectrallyresolved detection and corrects for effects of tissue optical absorption and scattering on the detected fluorescence signal to image the true fluorophore concentration. Pre-clinical studies in rodent glioma models using multiple fluorophores (PpIX, fluorescein) and clinical studies demonstrate improved residual tumor tissue detection. This quantitative, spectrally-resolved technique opens the door to simultaneous image-guided surgery of multiple fluorophores in the visible and near infrared.

The overall objective of the research is to investigate the utility of photochemical internalization for the enhanced nonviral transfection of genes into cells. We have examined, in detail, the evaluation of photochemical internalization (PCI) as a method for the non-viral introduction of the tumor suppressor gene PTEN and the PCI mediated transfection of the cytosine deaminase (CD) pro drug activating gene into glioma cell monolayers and multi-cell tumor spheroids. Expression of the CD gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic metabolite, 5-fluorouracil (5-FU).

We present work on a laser system operating in the near- and mid-IR spectral regions, having output characteristics designed to be optimal for cutting various tissue types. We provide a brief overview of laser-tissue interactions and the importance of controlling certain properties of the light beam. We describe the principle of operation of the laser system, which is generally based on a wavelength-tunable alexandrite laser oscillator/amplifier, and multiple Raman conversion stages. This configuration provides robust access to the mid-IR spectral region at wavelengths, pulse energies, pulse durations, and repetition rates that are attractive for neurosurgical applications. We summarize results for ultra-precise selective cutting of nerve sheaths and retinas with little collateral damage; this has applications in procedures such as optic-nerve-sheath fenestration and possible spinal repair. We also report results for cutting cornea, and dermal tissues.

Objective: Improvement of the clinical outcome of glioblastoma (GBM) patients by employment of fluorescence and photosensitization on the basis of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX). Methods: In this report the focus is laid on the use of tumor selective PpIX fluorescence for stereotactic biopsy sampling and intra-operative treatment monitoring. In addition, our current concept for treatment planning is presented. For stereotactic interstitial photodynamic therapy (iPDT), radial diffusers were implanted into the contrast enhancing tumor volume. Spectroscopic measurements of laser light transmission and fluorescence between adjacent fibers were performed prior, during and post PDT. Results: PpIX concentrations in primary glioblastoma tissue show high intra- and inter-patient variability, but are usually sufficient for an effective PDT. During individual treatment attempts with 5-ALA based GBM-iPDT, transmission and fluorescence measurements between radial diffusers gave the following results: 1. In some cases, transmission after PDT is considerably reduced compared to the value before PDT, which may be attributable to a depletion of oxygenated hemoglobin and/or diffuse bleeding. 2. PpIX fluorescence is efficiently photobleached during PDT in all cases. Conclusion: iPDT with assessment of PpIX fluorescence and photobleaching is a promising treatment option. Individualization of treatment parameters appears to bear a potential to further improve clinical outcomes.

PDT has been shown to be most effective at low fluence rates. Many radionuclides used for both diagnostic and therapeutic purposes produce measurable amounts of visible radiation when they decay via the Cerenkov effect which occurs when a charged particle travels faster in a dielectric medium than the speed of light in that medium. Cerenkov radiation from radiopharmaceuticals could serve as a source of extended duration, low level “internal” light, to mediate PDT, with the ultimate goals of overcoming some its current limitations. Using laser light, we are exploring the effects of fluence rates that could be generated by Cerenkov radiation on PDT efficacy. ALA or TPPS2a mediated PDT of rat gliomas monolayers or multicell spheroids ( F98, C6) was performed with 410 nm laser light exposure over an extended period of 24-96hrs. Photosensitizers were delivered either as a bolus or continuously with light exposure. At fluence rate of 20μW/cm² effective PDT was obtained as measured by decrease in cell viability or inhibition of spheroid growth. PDT is effective at ultra low fluence rates if given over long time periods. No lower threshold has been ascertained. Since the half-life of ⁹⁰Y, a radionuclide with a high Cherenkov yield is 64 hrs it is a good candidate to supply sufficient light activation for PDT. The combination of radionuclide and photodynamic therapies could improve the effectiveness of cancer treatment by exploiting synergies between these two modalities.

Identifying directional influences in neural circuits from functional near infrared spectroscopy (fNIRS) recordings presents one of the main challenges for understanding brain dynamics. In this study a new strategy that combines Granger causality mapping (GCM) and independent component analysis (ICA) is proposed to reveal complex neural network dynamics underlying cognitive processes with fNIRS measurements. The GCM-ICA algorithm implements the following two procedures: (i) extraction of the region of interests (ROIs) of cortical activations by ICA, and (ii) estimation of the direct causal influences in local brain networks using Granger causality among voxels of ROIs. Our results show the use of GCM in conjunction with ICA is able to effectively capture the brain network dynamics in time-frequency domain with significantly reduced computational cost. We thus suggest that the GCM-ICA technique is a potentially valuable tool that could be used for the investigation of directional causality influences of brain network dynamics in biophotonics fields.

We applied an orthogonal diffuse reflectance spectroscopy (o-DRS) to assess brain physiology following closed head injury (CHI). CHI was induced in anesthetized male mice by weight-drop device using ~50gram cylindrical metal falling from a height of 90 cm onto the intact scalp. A total of twenty-six mice were used in the experiments divided randomly into three groups as follows: Group 1 (n=11) consisted of injured mice monitored for 1 hour every 10 minutes. Group 2 (n=10) were the control mice not experience CHI. Group 3 (n=5) consisted of injured mice monitored every minute up to 20 minutes. Measurement of optical quantities of brain tissue (absorption and reduced scattering coefficients) in the near-infrared window from 650 to 1000 nm were carried out by employing different source-detector distances and locations to provide depth sensitivity. With respect to baseline, we found difference in brain hemodynamic properties following injury. In addition, o-DRS successfully evaluate the structural variations likely from evolving cerebral edema throughout exploring the scattering spectral shape.

To understand the pathophysiology of ischemic stroke, in vivo imaging of the brain tissue viability and related spreading depolarization is crucial. In the infarct core, impairment of energy metabolism causes anoxic depolarization (AD), which considerably increases energy consumption, accelerating irreversible neuronal damage. In the peri-infarct penumbra region, where tissue is still reversible despite limited blood flow, peri-infarct depolarization (PID) occurs, exacerbating energy deficit and hence expanding the infarct area. We previously showed that light-scattering signal, which is sensitive to cellular/subcellular structural integrity, was correlated with AD and brain tissue viability in a rat hypoxia-reoxygenation model. In the present study, we performed transcranial NIR diffuse reflectance imaging of the rat brain during middle cerebral artery (MCA) occlusion and examined whether the infarct core and PIDs can be detected. Immediately after occluding the left MCA, light scattering started to increase focally in the occlusion site and a bright region was generated near the occlusion site and spread over the left entire cortex, which was followed by a dark region, showing the occurrence of PID. The PID was generated repetitively and the number of times of occurrence in a rat ranged from four to ten within 1 hour after occlusion (n=4). The scattering increase in the occlusion site was irreversible and the area with increased scattering expanded with increasing the number of PIDs, indicating an expansion of the infarct core. These results suggest the usefulness of NIR diffuse reflectance signal to visualize spatiotemporal changes in the infarct area and PIDs.

Green fluorescent materials such as Green Fluorescence Protein (GFP) and fluorescein are often used for observing neural activities. Thus, it is important to observe the fluorescence in a freely moving state in order to understand neural activities corresponding to behaviors. In this work, we developed an implantable CMOS imaging device for in-vivo green fluorescence imaging with efficient excitation light rejection using a combination of absorption filters. An interference filter is usually used for a fluorescence microscope in order to achieve high fluorescence imaging sensitivity. However, in the case of the implantable device, interference filters are not suitable because their transmission spectra depend on incident angle. To solve this problem we used two kinds of absorption filters that do not have angle dependence. An absorption filter consisting of yellow dye (VARYFAST YELLOW 3150) was coated on the pixel array of an image sensor. The rejection ratio of ideal excitation light (490 nm) against green fluorescence (510 nm) was 99.66%. However, the blue LED as an excitation light source has a broad emission spectrum and its intensity at 510 nm is 2.2 x 10^-2 times the emission peak intensity. By coating LEDs with the emission absorption filters, the intensity of the unwanted component of the excitation light was reduced to 1.4 x 10^-4. Using the combination of absorption filters, we achieved excitation light transmittance of 10^-5 onto the image sensor. It is expected that high-sensitivity green fluorescence imaging of neural activities in a freely moving mouse will be possible by using this technology.

We investigate a method to estimate the spectral images of reduced scattering coefficients and the absorption coefficients of in vivo exposed brain tissues in the range from visible to near-infrared wavelength (500-760 nm) based on diffuse reflectance spectroscopy using a digital RGB camera. In the proposed method, the multi-spectral reflectance images of in vivo exposed brain are reconstructed from the digital red, green blue images using the Wiener estimation algorithm. The Monte Carlo simulation-based multiple regression analysis for the absorbance spectra is then used to specify the absorption and scattering parameters of brain tissue. In this analysis, the concentration of oxygenated hemoglobin and that of deoxygenated hemoglobin are estimated as the absorption parameters whereas the scattering amplitude a and the scattering power b in the expression of μ_s'=aλ^-b as the scattering parameters, respectively. The spectra of absorption and reduced scattering coefficients are reconstructed from the absorption and scattering parameters, and finally, the spectral images of absorption and reduced scattering coefficients are estimated. The estimated images of absorption coefficients were dominated by the spectral characteristics of hemoglobin. The estimated spectral images of reduced scattering coefficients showed a broad scattering spectrum, exhibiting larger magnitude at shorter wavelengths, corresponding to the typical spectrum of brain tissue published in the literature. In vivo experiments with exposed brain of rats during CSD confirmed the possibility of the method to evaluate both hemodynamics and changes in tissue morphology due to electrical depolarization.

Using near-infrared time-resolved spectroscopy (TRS), we measured the human head in transmittance mode to obtain the optical properties, tissue oxygenation, and hemodynamics of deep brain tissues in 50 healthy adult volunteers. The right ear canal was irradiated with 3-wavelengths of pulsed light (760, 795, and 835nm), and the photons passing through the human head were collected at the left ear canal. Optical signals with sufficient intensity could be obtained from 46 of the 50 volunteers. By analyzing the temporal profiles based on the photon diffusion theory, we successfully obtained absorption coefficients for each wavelength. The levels of oxygenated hemoglobin (HbO₂), deoxygenated hemoglobin (Hb), total hemoglobin (tHb), and tissue oxygen saturation (SO₂) were then determined by referring to the hemoglobin spectroscopic data. Compared with the SO₂ values for the forehead measurements in reflectance mode, the SO₂ values of the transmittance measurements of the human head were approximately 10% lower, and tHb values of the transmittance measurements were always lower than those of the forehead reflectance measurements. Moreover, the level of hemoglobin and the SO₂ were strongly correlated between the human head measurements in transmittance mode and the forehead measurements in the reflectance mode, respectively. These results demonstrated a potential application of this TRS system in examining deep brain tissues of humans.

The low frequency oscillation (LFO) around 0.1 Hz has been observed recently in cerebral hemodynamic signals during rest/sleep, enhanced breathing, and head- up-tilting, showing that cerebral autoregulation can be accessed by LFOs. However, many brain function researches require direct measurement of LFOs during specified brain function activities. This pilot study explored using near-infrared spectroscopy/imaging (NIRS) to noninvasively and simultaneously detect LFOs of prefrontal cerebral hemodynamics (i.e., oxygenated/deoxygenated/total hemoglobin concentration: △[oxy-Hb]/ △[deoxy-Hb]/ △[tot-Hb]) during N-back visual verbal working memory task. The LFOs were extracted from the measured variables using power spectral analysis. We found the brain activation sites struck clear LFOs while other sites did not. The LFO of △[deoxy-Hb] acted as a negative pike and ranged in (0.05, 0.1) Hz, while LFOs of △[oxy-Hb] and △[tot-Hb] acted as a positive pike and ranged in (0.1, 0.15) Hz. The amplitude difference and frequency lag between △[deoxy-Hb] and △[oxy-Hb]/ △[tot-Hb] produced a more focused and sensitive activation map compare to hemodynamic amplitude-quantified activation maps. This study observed LFOs in brain activities and showed strong potential of LFOs in accessing brain functions.

While most commercially available functional near-infrared spectroscopy (fNIRS) systems employ optical fibers for both the measurement optode and the transmission cable for optical signals, their material inflexibility presents some problems in stable optode fixation to the head surface and adequate cable lining to the main system. In practice, mechanical fluctuations of optical fibers in fNIRS measurement often lead to motion artifacts in the signals. A few fiberless fNIRS systems are available and equipped with light sources and detectors that directly adhere to the scalp surface. However, their shapes and detection sensitivities are not suitable for usage on a hair-covered head. Based on the commercial fiber-less fNIRS system OEG-16 (Spectratech Inc., Japan), we developed a new source-detector unit that was designed with LEDs for enhanced illumination, avalanche photodiodes instead of photodiodes, and a new holder system. The electrical circuits of the system were modified after the design. By simultaneous implementation of multidistance fNIRS measurement and hemodynamic modality separation on conventional fNIRS data at the bilateral parietal area during single-sided motor tasks, significant functional signals were observed only at the position contralateral to the side of movement. This is the first report describing a fiber-less fNIRS system that can detect functional signals on a hair-covered head. We believe this fiber-less system will improve the utility of fNIRS, particularly in less restraining conditions.

Similar to blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI), functional nearinfrared spectroscopy (fNIRS) observes regional hemodynamic responses associated with neuronal activation. However, the conventional criteria for detecting true positive fNIRS and fMRI signals appear to be based on different understandings of cerebral hemodynamics. Considerable numbers of fNIRS studies have ascribed the increase in oxygenated hemoglobin to a typical sign of functional activation, whereas the corresponding BOLD signal in fMRI directly correlates with a decrease in deoxygenated hemoglobin. This inconsistency requires solution through the simultaneous measurements of fNIRS and fMRI. In practice, however, there remain several technical problems associated with conducting simultaneous measurements with high reproducibility. One issue is the precise spatial registration of NIRS optodes in MR images. We prepared marker containers of an annular shape that can be coaxially fixed to the optode. Liquid paraffin with α-tocopheryl acetate, which exhibits a bright contrast in T1-weighted MR images of human heads, was solidified in each container by adding higher fatty acid. A subject wearing the marker-fixed optodes at parietal area participated in preliminary fNIRS and fMRI experiments; the subject was instructed to execute single-sided hand finger tapping. The positions showed that deoxygenated hemoglobin decreases in fNIRS coincided with the BOLD-positive region in fMRI. The prepared marker is chemically stable and repetitively usable. We believe that this simple method contributes precision to the co-registration of fNIRS and fMRI.

Imaging brain circuits is the basis for us to understand brain function and dysfunction. However, imaging axon at micrometer resolution while tracing the centimeter-scale axon projection across the whole-brain is still challenging. Here, we developed a fluorescence micro-optical sectioning tomography (fMOST) imaging system based on confocal fluorescence imaging scheme that can obtain whole brain image stack for visualizing brain circuits at neurite level. We use confocal detection to remove fluorescence background to clearly see one single neurite and use acoustical optical deflector (AOD), an inertia-free beam scanner to realize fast and prolonged stable imaging. We had acquired several complete datasets of whole-mouse brain at a one-micron voxel resolution. Based on these datasets, the uninterrupted tracing of brain-wide, long-distance axonal projections was demonstrated for the first time using a systematic reconstruction and annotation pipeline. Our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.

The efficacy of gold-silica nanoshells (AuNS) and gold nanorods (AuNR) for photothermal therapy was investigated in an in vitro system consisting of hybrid murine macrophage/human glioma spheroids. Macrophages were used as delivery vectors for the nanoparticles. Hybrid spheroids were formed via centrifugation of human glioma cells and nanoparticleloaded macrophages. Forty-eight hours post-centrifugation, the resultant 400 μm dia. spheroids were exposed to 808 nm laser light for 10 min. at irradiances ranging from 2 - 28 W cm^-2. Treatment efficacy was evaluated from spheroid growth kinetics over a 14-day period. AuNS were shown to have greater efficacy compared to AuNR. For example, hybrid spheroids consisting of a 5:1 ratio of glioma cells to AuNS-loaded macrophages exhibited significant growth inhibition when subjected to irradiances of 7 W cm^-2. In contrast, no growth inhibition was observed for the AuNR-macrophage hybrid spheroids, even at the highest irradiance investigated (28 W cm^-2). Growth inhibition was observed at 28 W cm^-2 when the nanorod concentration was increased, i.e., by forming hybrid spheroids with a 2:1 ratio of glioma cells to macrophages.

To study neuronal functions in brain, we developed a higher resolution type fiber-coupled microscope (FCM), and measured the activity-dependent fluorescence intensity of the excitable cells over time. FCM was constructed by combining a fluorescence microscope with the high density type of fiber bundle, which consisted of 1.5 x 10⁴ unit fiber in the assemble less than 0.5 mm tip. The spatial resolution was calculated to be 2.4 mm with the 5 mm focal depth. The activity-dependent Ca signals were detectable in each cell of either the pancreatic spheroids or the brain slices. The present FCM is very promising for detailed studies with the live imaging of signal molecules in the body at a single cell level.

We measured hemodynamic activity of the prefrontal cortex (PFC) during a Chinese color-word matching Stroop task using a homemade continuous-wave NIRS system. Two probes were placed separately over the left and the right PFC. Wavelet transform coherence (WTC) analysis was employed to calculate coherences between all channels of the same probe pairwise to obtain the intrahemispheric functional connectivity for each side of the PFC. The intrahemispheric functional connectivities in both sides of PFC were stronger during the incongruent task compared to that of the neutral task, but only the left intrahemispheric functional connectivity showed a significant Stroop effect. In addition to the Stroop effect, for the incongruent or the neutral task, there was also a leftward lateralization. The results indicate that, compared with traditional activation, NIRS-based connectivity is more sensitive for identifying hemispheric lateralization.

Near-infrared spectroscopy (NIRS) is a non-invasive tool to measure real-time tissue oxygenation in the brain. In an invasive animal experiment we were able to directly compare non-invasive NIRS measurements on the skull with invasive measurements directly on the brain dura matter. We used a broad-band, continuous-wave hyper-spectral approach to measure tissue oxygenation in the brain of pigs under the conditions of cardiac arrest, cardiopulmonary resuscitation (CPR), and defibrillation. An additional purpose of this research was to find a correlation between mortality due to cardiac arrest and inadequacy of the tissue perfusion during attempts at resuscitation. Using this technique we measured the changes in concentrations of oxy-hemoglobin [HbO2] and deoxy-hemoglobin [HHb] to quantify the tissue oxygenation in the brain. We also extracted cytochrome c oxidase changes Δ[Cyt-Ox] under the same conditions to determine increase or decrease in cerebral oxygen delivery. In this paper we proved that applying CPR, [HbO2] concentration and tissue oxygenation in the brain increase while [HHb] concentration decreases which was not possible using other measurement techniques. We also discovered a similar trend in changes of both [Cyt-Ox] concentration and tissue oxygen saturation (StO2). Both invasive and non-invasive measurements showed similar results.

There is a controversy, to which extend cochlear stimulation with near infrared laser pulses at a wavelength of 1860 nm is based on optoacoustic stimulation of intact hair cells or -in contrast- is based on direct stimulation of the nerve cells in absence of functional hair cells. Thermal and stress confinement conditions apply, because of the pulse duration range (5 ns, 10 μs-20 ms) of the two lasers used. The dependency of the signal characteristics on pulse peak power and pulse duration was investigated in this study. The compound action potential (CAP) was measured during stimulation of the cochlea of four anaesthetized guinea pigs, which were hearing at first and afterwards acutely deafened using intracochlear neomycin-rinsing. For comparison hydrophone measurements in a water tank were performed to investigate the optoacoustic signals at different laser interaction regimes. With rising pulse peak power CAPs of the hearing animals showed first a threshold, then a positively correlated and finally a saturating dependency. CAPs also showed distinct responses at laser onset and offset separated with the pulse duration. At pulse durations shorter than physiological response times the signals merged. Basically the same signal characteristics were observed in the optoacoustic hydrophone measurements, scaled with the sensitivity and response time of the hydrophone. Taking together the qualitative correspondence in the signal response and the absence of any CAPs in deafened animals our results speak in favor of an optoacoustic stimulation of intact hair cells rather than a direct stimulation of nerve cells.

Infrared neural stimulation (INS) has been used in the past to evoke neural activity from hearing and partially deaf animals. All the responses were excitatory. In Aplysia californica, Duke and coworkers demonstrated that INS also inhibits neural responses [1], which similar observations were made in the vestibular system [2, 3]. In deaf white cats that have cochleae with largely reduced spiral ganglion neuron counts and a significant degeneration of the organ of Corti, no cochlear compound action potentials could be observed during INS alone. However, the combined electrical and optical stimulation demonstrated inhibitory responses during irradiation with infrared light.

Spatial selective infrared neural stimulation has potential to improve neural prostheses, including cochlear implants. The heating of a confined target volume depolarizes the cell membrane and results in an action potential. Tissue heating may also result in the generation of a stress relaxation wave causing mechanical stimulation of hair cells in the cochlea, creating an optoacoustic response. Data are presented that quantify the effect of an acoustical stimulus (noise masker) on the response obtained with INS in normal hearing, and chronic deaf animals. While in normal hearing animals an acoustic masker can reduce the response to INS, in chronic deaf animals this effect has not been detected. The responses to INS remain stable following the different degrees of cochlear damage.

Deficits in prefrontal function play a crucial role in compulsive cocaine use, which is a hallmark of addiction. Dysfunction of the prefrontal cortex might result from effects of cocaine on neurons as well as from disruption of cerebral blood vessels. However, the mechanisms underlying cocaine’s neurotoxic effects are not fully understood, partially due to technical limitations of current imaging techniques (e.g., PET, fMRI) to differentiate vascular from neuronal effects at sufficiently high temporal and spatial resolutions. We have recently developed a multimodal imaging platform which can simultaneously characterize the changes in cerebrovascular hemodynamics, hemoglobin oxygenation and intracellular calcium fluorescence for monitoring the effects of cocaine on the brain. Such a multimodality imaging technique (OFI) provides several uniquely important merits, including: 1) a large field-of-view, 2) high spatiotemporal resolutions, 3) quantitative 3D imaging of the cerebral blood flow (CBF) networks, 4) label-free imaging of hemodynamic changes, 5) separation of vascular compartments (e.g., arterial and venous vessels) and monitoring of cortical brain metabolic changes, 6) discrimination of cellular (neuronal) from vascular responses. These imaging features have been further advanced in combination with microprobes to form micro-OFI that allows quantification of drug effects on subcortical brain. In addition, our ultrahigh-resolution ODT (μODT) enables 3D microangiography and quantitative imaging of capillary CBF networks. These optical strategies have been used to investigate the effects of cocaine on brain physiology to facilitate the studies of brain functional changes induced by addictive substance to provide new insights into neurobiological effects of the drug on the brain.

Clinically it is important to image structures of brain at deeper areas with low invasions, for example, the pathological information is not obtained enough from the white matter. Preliminarily we have measured transmission images of rat brain using the short graded-index multimode fiber (SMMF) with the diameter of 140μm and length of 5mm. SMMF (core diameter, 100μm) was cut using a fiber cleaver and was fixed in a jig. Fiber lengths inside and outside jig were 3mm and 2mm, respectively. The jig was attached at the 20x objective lens. The conventional optical microscope was used to measure images. In basic characteristics, it was confirmed that the imaging conditions almost corresponded to calculations with the ray-transfer matrix and the spatial resolution was evaluated at about 4.4μm by measuring the test pattern. After euthanasia the rat parietal brain was excised with thickness around 1.5mm and was set on the slide glass. The tissue was illuminated through the slide glass by the bundle fiber with Halogen lamp. The tip of SMMF was inserted into the tissue by lifting the sample stage. The transmission image at each depth from 0.1mm to 1.53mm was measured. Around the depth of 1.45mm, granular structures with sizes of 4-5μm were recognized and corresponded to images by HE stained tissue. Total measurement time was within 2 hours. The feasibilities to image the depth of 5 mm with SMMF have been shown.

Multi-photon imaging provides valuable insights into the continuous reshaping of neuronal connectivity in live brain. We previously showed that single neuron or even single spine ablation can be achieved by laser-mediated dissection. Furthermore, single axonal branches can be dissected avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Here, we describe the procedure to address the structural plasticity of cerebellar climbing fibers by combining two-photon in vivo imaging with laser axotomy in a mouse model. This method is a powerful tool to study the basic mechanisms of axonal rewiring after single branch axotomy in vivo. In fact, despite the denervated area being very small, the injured axons consistently reshape the connectivity with surrounding neurons, as indicated by the increase in the turnover of synaptic boutons. In addition, time-lapse imaging reveals the sprouting of new branches from the injured axon. Newly formed branches with varicosities suggest the possible formation of synaptic contacts. Correlative light and electron microscopy revealed that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites.

We examined longitudinal changes of the neuro-glia-vascular unit during cerebral angiogenesis induced through chronic hypoxia in the adult mouse cortex. Tie2-GFP mice in which the vascular endothelial cells expressed green fluorescent proteins (GFP) were exposed to chronic hypoxia, while the spatiotemporal developments of the cortical capillary sprouts and the neighboring astrocytic remodeling were characterized with repeated two-photon microscopy. The capillary sprouts appeared at early phases of the hypoxia adaptation (1-2 weeks), while the morphological changes of the astrocytic soma and processes were not detected in this phase. In the later phases of the hypoxia adaptation (> 2 weeks), the capillary sprouts created a new connection with existing capillaries, and its neighboring astrocytes extended their processes to the newly-formed vessels. The findings show that morphological adaptation of the astrocytes follow the capillary development during the hypoxia adaptation, which indicate that the newly-formed vessels provoke cellular interactions with the neighboring astrocytes to strengthen the functional blood-brain barrier.

In obstructive sleep apnea syndrome (OSA) the periodic reduction or cessation of breathing due to narrowing or occlusion of the upper airway during sleep leads to daytime symptoms and increased cardiovascular risk, including stroke. The higher risk of stroke is related to the impairment in cerebral vascular autoregulation. Continuous positive airways pressure (CPAP) therapy at night is the most effective treatment for OSA. However, there is no suitable bedside monitoring method evaluating the treatment efficacy of CPAP therapy, especially to monitor the recovery of cerebral hemodynamics. NIRS is ideally suited for non-invasive monitoring the cerebral hemodynamics during sleep. In this study, we will for first time assess dynamic changes of cerebral hemodynamics during nocturnal CPAP therapy in 3 patients with OSA using NIRS. We found periodic oscillations in HbO2, HHb, tissue oxygenation index (TOI) and blood volume associated with periodic apnea events without CPAP in all OSA patients. These oscillations were gradually attenuated and finally eliminated with the stepwise increments of CPAP pressures. The oscillations were totally eliminated in blood volume earlier than in other hemodynamic parameters. These results suggested that 1) the cerebral hemodynamic oscillations induced by OSA events can effectively be attenuated by CPAP therapy, and 2) blood flow and blood volume recovered first during CPAP therapy, followed by the recovery of oxygen consumption. Our study suggested that NIRS is a useful tool to evaluate the efficacy of CPAP therapy in patients with OSA bedside and in real time.

Obstructive sleep apnea syndrome (OSA) and periodic limb movement in sleep syndrome (PLMS) are two common sleep disorders. Previous studies showed that OSA and PLMS share common features, such as increased cardio-vascular risk, both apnea events and limb movements occur periodically, they are usually associated with cortical arousals, and both of them can induce declines in peripheral oxygen saturation measured with pulse oximetry. However, the question whether apnea events and limb movements also show similar characteristics in cerebral hemodynamic and oxygenation has never been addressed. In this pilot study, we will first time compare the cerebral hemodynamic changes induced by apnea events and limb movements in patients with OSA (n=4) and PLMS (n=4) with NIRS. In patients with OSA, we found periodic oscillations in HbO₂, HHb, and blood volume induced by apnea/hypopnea events, HbO₂ and HHb showed reverse changing trends. By contrast, the periodic oscillations linked to limb movements were only found in HbO₂ and blood volume in patients with PLMS. These findings of different cerebral hemodynamics patterns between apnea events and limb movements may indicate different regulations of nervous system between these two sleep disorders.

Growth performance, behavior, and development of broilers reared under red, green, and blue monochromatic and/or multicolor LED-based illuminants is investigated. The lighting treatments were performed on a 24h lighting basis during six weeks. Monochromatic red(630 nm), green(520 nm), and blue(460 nm), and simultaneous blue-green, and whitelight housing illumination was employed. Bodyweight, food consumption, and behavior were monitored and compared amongst light treatments. The behavioral data showed that broilers reared under green lighting presented the lowest respiratory rate (87 mov. min^-1) while those under red lighting presented the highest (96 mov. min^-1). Results also showed that broilers under blue and/or green monochromatic illumination exhibited up to 6%, and 8.9 % increase in final bodyweight when compared to those under red or white-light, respectively. The highest feed intake, and lowest body weight gain was observed in broilers reared under blue and red illumination, respectively.

Blast-induced traumatic brain injury is a growing concern, but its underlying pathophysiology and mechanism are still unknown. Thus, study using an animal model is needed. We have been proposing the use of a laser-induced shock wave (LISW), whose energy is highly controllable and reproducible, to mimic blast-related injury. We previously observed the occurrence of spreading depolarization (SD) and prolonged hypoxemia in the rat brain exposed to an LISW. However, the relationship between these two events is unclear. In this study, we investigated the spatiotemporal characteristics of hypoxemia and SD to examine their correlation, for which multichannel fiber measurement and multispectral imaging of the diffuse reflectance were performed for the rat brain exposed to an LISW. We also quantified tissue oxygen saturation (StO₂) in the hypoxemic phase, which is associated with possible neuronal cell death, based on an inverse Monte Carlo simulation. Fiber measurement showed that the region of hypoxemia was expanding from the site of LISW application to the distant region over the brain; the speed of expansion was similar to that of the propagation speed of SD. Simulation showed that oxygen saturation was decreased by ~40%. Multispectral imaging showed that after LISW application, a vasodilatation occurred for ~1 min, which was followed by a long-lasting vasoconstriction. In the phase of vasoconstriction, StO₂ declined all over the field of view. These results indicate a strong correlation between SD and hypoxemia; the estimated StO₂ seems to be low enough to induce neuronal cell death.

The manipulation of genetically targeted neurons with light (optogenetics) continues to provide unprecedented avenues into studying the function of the mammalian brain. However, potential translation into the clinical arena faces a number of significant hurdles, foremost among them the need for insertion of optical fibers into the brain to deliver light to opsins expressed on neuronal membranes. In order to overcome these hardware-related problems, we have developed an alternative strategy for delivering light to opsins which does not involve fiber implants. Rather, the light is produced by a protein, luciferase, which oxidizes intravenously applied substrate, thereby emitting bioluminescence. In proof-ofprinciple studies employing a fusion protein of a light-generating luciferase to a light-sensing opsin (luminopsin), we showed that light emitted by Gaussia luciferase is indeed able to activate channelrhodopsin, allowing modulation of neuronal activity when expressed in cultured neurons. Here we assessed applicability of the concept in vivo in mice expressing luminopsins from viral vectors and from genetically engineered transgenes. The experiments demonstrate that intravenously applied substrate reaches neurons in the brain, causing the luciferase to produce bioluminescence which can be imaged in vivo, and that activation of channelrhodopsin by bioluminescence is sufficient to affect behavior. Further developments of such technology based on combining optogenetics with bioluminescence - i.e. combining lightsensing molecules with biologically produced light through luciferases - should bring optogenetics closer to clinical applications.

Fiber-optic based optical imaging is an emerging technique for studying brain activity in live animals. Here, we introduce a novel fluorescence fiber-optic microendoscopy approach to minimal invasively detect neural activities in a live mouse brain . The system uses a flexible endoscopic probe composed of a multi-core coherent fiber-bundle terminated with an approximately 1500-micron working distance objective lens. The fiber-optic neural interface is mounted on a 4-mm² cranial window enabling visualization of glial calcium transients from the same brain region for weeks. We evaluated the system performance through in vivo imaging of GCaMP3 fluorescence in transgenic headrestrained mice during locomotion.

Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

In "New Small Quantum Dots for Neuroscience," Paul Selvin (University of Illinois, Urbana-Champaign) notes how the details of synapsis activity in the brain involves chemical receptors that facilitate the creation of the electrical connection between two nerves. In order to understand the details of this neuroscience phenomenon you need to be able to "see" what is happening at the scale of these receptors, which is around 10 nanometers. This is smaller than the diffraction limit of normal microscopy and it takes place on a 3 dimensional structure. Selvin describes the development of small quantum dots (on the order of 6-9 microns) that are surface-sensitized to interact with the receptors. This allows the application of photo-activated localized microscopy (PALM), a superresolution microscopy that can be scanned through focus to develop a 3D map on a scale that is the same size as the emitter, which in this case are the small quantum dots. The quantum dots are stable in time and provide access to the receptors which allows the imaging of the interactions taking place at the synoptic level.

The study of the brain's plasticity, or its ability to change, helps to reveal how it works. The secrets of brain activity and its control of motion are hidden in the structures, the functionality and the morphology of the physical brain, notes Francesco Pavone of the European Laboratory for Non-Linear Spectroscopy (LENS) in his presentation,"Journey into the Brain: from Single Synapse to Whole Brain Anatomy by Correlative Microscopy." We know synapses are formed through chemical interactions and electrical connections are made, Pavone says. But in order to understand the process we must examine the brain at several different scales. A cadre of optical methods such as correlative microscopy, optical manipulation, 3D tomography, confocal light sheet microscopy, and multimodal camera imaging are used to examine the brain at highly localized regions but at the multiple scales to reveal these inner workings.