This PDF file contains the front matter associated with SPIE Proceedings Volume 9305, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

The pathology of multiple sclerosis involves the gray and white matter regions of the brain and spinal cord often characterized by various combinations of demyelination, inflammatory infiltration, axonal degeneration, and later gliosis in chronic lesions. While acute and chronic white matter lesions are well characterized and easily identified, evidence indicates that the CNS of MS patients may be globally altered, with subtle abnormalities found in grossly normal appearing white matter (NAWM) with histochemical stains and magnetic resonance imaging only indicating a general alteration in tissue composition at best. Thus, the prototypical acute inflammatory lesion may merely represent the most obvious manifestation of a chronic widespread involvement of the CNS, which is difficult to examine reliably. The current study deals with the microstructure and biochemistry of demyelination, remyelination and axonal loss in various regions in post-mortem human MS brain, especially NAWM areas around more typical acute and chronic lesions. The myelin sheath, neuroglia and perivascular spaces were investigated through changes in the intrinsic molecular vibrational signatures of lipid biochemistry using a novel, label-free Coherent anti-Stokes Raman Scattering (CARS) microscope. The biochemistry of myelin lipids can be probed by detecting subtle changes to phospholipids and the intra-molecular disorder of their fatty acid acyl chains, various oxidation products and general protein contributions. NAWM regions surrounding pathological MS lesions were shown to reveal abnormalities despite morphological classifications indicating otherwise. CARS data were correlated with immunohistochemical stains and lipophilic dyes. Spectral data were analyzed using a unique non-linear algorithm, which allows quantification and classification through gated parameters and displayed through bivariate histograms. Our CARS microscopy system provides high-resolution, detailed morphological and unique biochemical information regarding CNS pathology in human MS examples and may be applicable to a broad range of other white matter centric neurological disorders.

This study presents the design of a system used to monitor laser ablation in real-time using Optical Coherence Tomography (OCT). The design of the system involves a high-powered fiber laser (wavelength of 1064nm, 1kW peak power) being built directly into the sample arm of the OCT system (center wavelength 1310). It is shown that the OCT laser light and subsequent backscatter pass relatively unaffected through the fiber laser. Initial results are presented showing monitoring of the ablation process at a single point in real time using m-mode imaging.

Optical Coherence Tomography (OCT) has extensive potential for producing clinical impact in the field of neurological diseases. A neurosurgical OCT hand-held forward viewing probe in Bayonet shape has been developed. In this study, we test the feasibility of integrating this imaging probe with modern navigation technology for guidance and monitoring of skull base surgery. Cadaver heads were used to simulate relevant surgical approaches for treatment of sellar, parasellar and skull base pathology. A high-resolution 3D CT scan was performed on the cadaver head to provide baseline data for navigation. The cadaver head was mounted on existing 3- or 4-point fixation systems. Tracking markers were attached to the OCT probe and the surgeon-probe-OCT interface was calibrated. 2D OCT images were shown in real time together with the optical tracking images to the surgeon during surgery. The intraoperative video and multimodality imaging data set, consisting of real time OCT images, OCT probe location registered to neurosurgical navigation were assessed. The integration of intraoperative OCT imaging with navigation technology provides the surgeon with updated image information, which is important to deal with tissue shifts and deformations during surgery. Preliminary results demonstrate that the clinical neurosurgical navigation system can provide the hand held OCT probe gross anatomical localization. The near-histological imaging resolution of intraoperative OCT can improve the identification of microstructural/morphology differences. The OCT imaging data, combined with the neurosurgical navigation tracking has the potential to improve image interpretation, precision and accuracy of the therapeutic procedure.

The use of gas assistance in laser machining hard materials is well established in manufacturing but not in the context of surgery. Laser cutting of osseous tissue in the context of neurosurgery can benefit from gas-assist but requires an understanding of flow and pressure effects to minimize neural tissue damage. In this study we acquire volumetric flow rates through a gas nozzle on the spinal cord, with dura and without dura.

Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

Background
Glioblastoma is a high-grade cerebral tumor with local recurrence and poor outcome. Photodynamic therapy (PDT) is a local treatment based on the light activation of a photosensitizer (PS) in the presence of oxygen to form cytotoxic species. Fractionation of light delivery may enhance treatment efficiency by restoring tissue oxygenation.

Objectives
To evaluate the efficiency of light fractionation using MRI imaging, including diffusion and perfusion, compared to histological data.

Materials and Methods
Thirty-nine “Nude” rats were grafted with human U87 cells into the right putamen. After PS precursor intake (5-ALA), an optic fiber was introduced into the tumor. The rats were randomized in three groups: without illumination, with monofractionated illumination and the third one with multifractionated light. Treatment effects were assessed with early MRI including diffusion and perfusion sequences. The animals were eventually sacrificed to perform brain histology.

Results
On MRI, we observed elevated diffusion values in the center of the tumor among treated animals, especially in multifractionated group. Perfusion decreased around the treatment site, all the more in the multifractionated group. Histology confirmed our MRI findings, with a more extensive necrosis and associated with a rarified angiogenic network in the treatment area, after multifractionated PDT. However, we observed more surrounding edema and neovascularization in the peripheral ring after multifractionated PDT.

Conclusion
Fractionated interstitial PDT induced specific tumoral lesions. The multifractionated scheme was more efficient, inducing increased tumoral necrosis, but it also caused significant peripheral edema and neovascularization. Diffusion and perfusion MRI imaging were able to predict the histological lesions.

Hyperthermia has been shown to enhance the effects of chemotherapeutic agents in a wide variety of cancers. The purpose of this study was to investigate the combined effects of a number of commonly used chemotherapeutic drugs (bleomycin, doxorubicin and cisplatin) with photothermal therapy (PTT)-induced hyperthermia in an in vitro system consisting of human head and neck squamous carcinoma cells and murine lymphocytic monocytes which were used as delivery vehicles for gold-silica nanoshells (AuNS). PTT was accomplished via near infra-red (NIR) irradiation of AuNS. The results showed that PTT combined with cisplatin resulted in only a mild degree of synergism while additive effects were observed for concurrent treatments of PTT and doxorubicin and PTT and bleomycin.

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Multispectral imaging has received significant attention over the last decade as it integrates spectroscopy, imaging, tomography analysis concurrently to acquire both spatial and spectral information from biological tissue. In the present study, a multispectral setup based on projection of structured illumination at several near-infrared wavelengths and at different spatial frequencies is applied to quantitatively assess brain function before, during, and after the onset of traumatic brain injury in an intact mouse brain (n=5). For the production of head injury, we used the weight drop method where weight of a cylindrical metallic rod falling along a metal tube strikes the mouse’s head. Structured light was projected onto the scalp surface and diffuse reflected light was recorded by a CCD camera positioned perpendicular to the mouse head. Following data analysis, we were able to concurrently show a series of hemodynamic and morphologic changes over time including higher deoxyhemoglobin, reduction in oxygen saturation, cell swelling, etc., in comparison with baseline measurements. Overall, results demonstrates the capability of multispectral imaging based structured illumination to detect and map of brain tissue optical and physiological properties following brain injury in a simple noninvasive and noncontact manner.

Diffuse reflectance spectroscopy using fiber optic probe is one of most promising technique for evaluating optical properties of biological tissue. We present a method determining the reduced scattering coefficients μ_s’, the absorption coefficients μ a, and tissue oxygen saturation StO₂ of in vivo brain tissue using single reflectance fiber probe with two source-collector geometries. In the present study, we performed in vivo recordings of diffuse reflectance spectra and the electrophysiological signals for exposed brain of rats during normoxia, hyperoxia, hypoxia, and anoxia. The time courses of μ_a in the range from 500 to 584 nm and StO₂ indicated the hemodynamic change in cerebral cortex. Time courses of μ_s’ are well correlated with those of μ_a in the range from 530 to 570 nm, which also reflect the scattering by red blood cells. On the other hand, a fast decrease in μ_s’ at 800 nm were observed after the respiratory arrest and it synchronized with the negative deflection of the extracellular DC potential. It is said that the DC shift coincident with a rise in extracellular potassium and can evoke cell deformation generated by water movement between intracellular and extracellular compartments, and hence the light scattering by tissue. Therefore, the decrease in μ_s’ at 800 after the respiratory arrest is indicative of changes in light scattering by tissue. The results in this study indicate potential of the method to evaluate the pathophysiological and loss of tissue viability in brain tissue.

Sensorimotor cortex plasticity induced by constraint-induced movement therapy (CIMT) in six children (10.2 ± 2.1 years old) with hemiplegic cerebral palsy (CP) was assessed by functional near-infrared spectroscopy (fNIRS). The activation laterality index and time-to-peak/duration during a finger tapping task were quantified before, immediately after, and six months after CIMT. Five age-matched healthy children (9.8 ± 1.3 years old) were also imaged at the same time points to provide comparative activation metrics for normal controls. In children with CP the activation time-to-peak/duration for all sensorimotor centers displayed significant normalization immediately after CIMT that persisted six months later. In contrast to this longer term improvement in localized activation response, the laterality index that depended on communication between sensorimotor centers improved immediately after CIMT, but relapsed six months later.

Carotid atherosclerosis is a critical medical concern that can lead to ischemic stroke. Local hemodynamic patterns have also been associated with the development of atherosclerosis, particularly in regions with disturbed flow patterns such as bifurcations. Traditionally, this disease was treated using carotid endarterectomy, however recently there is an increasing trend of carotid artery stenting due to its minimally invasive nature. It is well known that this interventional technique creates changes in vasculature geometry and hemodynamic patterns due to the interaction of stent struts with arterial lumen, and is associated with complications such as distal emboli and restenosis. Currently, there is no standard imaging technique to evaluate regional hemodynamic patterns found in stented vessels. Doppler optical coherence tomography (DOCT) provides an opportunity to identify in vivo hemodynamic changes in vasculature using high-resolution imaging. In this study, blood flow profiles were examined at the bifurcation junction in the internal carotid artery (ICA) in a porcine model following stent deployment. Doppler imaging was further conducted using pulsatile flow in a phantom model, and then compared to computational fluid dynamics (CFD) simulation of a virtual bifurcation to assist with the interpretation of emphin vivo results.

Spinal surgery is particularly challenging for surgeons, requiring a high level of expertise and precision without being able to see beyond the surface of the bone. Accurate insertion of pedicle screws is critical considering perforation of the pedicle can result in profound clinical consequences including spinal cord, nerve root, arterial injury, neurological deficits, chronic pain, and/or failed back syndrome. Various navigation systems have been designed to guide pedicle screw fixation. Computed tomography (CT)-based image guided navigation systems increase the accuracy of screw placement allowing for 3- dimensional visualization of the spinal anatomy. Current localization techniques require extensive preparation and introduce spatial deviations. Use of near infrared (NIR) optical tracking allows for realtime navigation of the surgery by utilizing spectral domain multiplexing of light, greatly enhancing the surgeon’s situation awareness in the operating room. While the incidence of pedicle screw perforation and complications have been significantly reduced with the introduction of modern navigational technologies, some error exists. Several parameters have been suggested including fiducial localization and registration error, target registration error, and angular deviation. However, many of these techniques quantify error using the pre-operative CT and an intra-operative screenshot without assessing the true screw trajectory. In this study we quantified in-vivo error by comparing the true screw trajectory to the intra-operative trajectory. Pre- and post- operative CT as well as intra-operative screenshots were obtained for a cohort of patients undergoing spinal surgery. We quantified entry point error and angular deviation in the axial and sagittal planes.

The simultaneous visualization, identification and targeting of neurons during patch clamp-mediated electrophysiological recordings is a basic technique in neuroscience, yet it is often complicated by the inability to visualize the pipette tip, particularly in deep brain tissue. Here we demonstrate a novel approach in which fluorescent quantum dot probes are used to coat pipettes prior to their use. The strong two-photon absorption cross sections of the quantum dots afford robust contrast at significantly deeper penetration depths than current methods allow. We demonstrate the utility of this technique in multiple recording formats both in vitro and in vivo where imaging of the pipettes is achieved at remarkable depths (up to 800 microns). Notably, minimal perturbation of cellular physiology is observed over the hours-long time course of neuronal recordings. We discuss our results within the context of the role that quantum dot nanoprobes may play in understanding neuronal cell physiology.

Simultaneous measurement of hemodynamics is of great importance to evaluate the brain functional changes induced by brain diseases such as drug addiction. Previously, we developed a multimodal-imaging platform (OFI) which combined laser speckle contrast imaging with multi-wavelength imaging to simultaneously characterize the changes in cerebral blood flow (CBF), oxygenated- and deoxygenated- hemoglobin (HbO and HbR) from animal brain. Recently, we upgraded our OFI system that enables detection of hemodynamic changes in response to forepaw electrical stimulation to study potential brain activity changes elicited by cocaine. The improvement includes 1) high sensitivity to detect the cortical response to single forepaw electrical stimulation; 2) high temporal resolution (i.e., 16Hz/channel) to resolve dynamic variations in drug-delivery study; 3) high spatial resolution to separate the stimulation-evoked hemodynamic changes in vascular compartments from those in tissue. The system was validated by imaging the hemodynamic responses to the forepaw-stimulations in the somatosensory cortex of cocaine-treated rats. The stimulations and acquisitions were conducted every 2min over 40min, i.e., from 10min before (baseline) to 30min after cocaine challenge. Our results show that the HbO response decreased first (at ~4min) followed by the decrease of HbR response (at ~6min) after cocaine, and both did not fully recovered for over 30min. Interestingly, while CBF decreased at 4min, it partially recovered at 18min after cocaine administration. The results indicate the heterogeneity of cocaine’s effects on vasculature and tissue metabolism, demonstrating the unique capability of optical imaging for brain functional studies.

Light scattering inside biological tissue is a limitation for large volumes imaging with microscopic resolution. Based on refractive index matching, different approaches have been developed to reduce scattering in fixed tissue. High refractive index organic solvents and water-based optical clearing agents, such as Sca/e, SeeDB and CUBIC have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability to large volumes. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging blocks of dysplastic human brain transformed with CLARITY, immunostained and cleared with the TDE. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.

Brain functional connectivity is mapped using spontaneous low-frequency oscillations (LFOs) in blood-oxygen-leveldependent (BOLD) signals using fMRI. However, the origin of spontaneous BOLD oscillations remains elusive. Specifically, the coupling of regional hemodynamic LFOs to neuronal activity in a resting brain is rarely examined directly. Here we present a method based on instantaneous-frequency (IF) analysis to detect regional LFOs of cerebral blood flow (CBF) along with local-field potential (LFP) changes of neurons in resting state to study neurovascular coupling. CBF and LFP were simultaneously acquired using laser Doppler flowmetry (LDF) and electroencephalography in the rat’s somatosensory cortex with high temporal resolution (i.e., 20Hz for CBF and 2kHz for LDF, respectively). Instead of fast Fourier transform analysis, a peak-detection algorithm was used to define the LFP activities and CBF spontaneous oscillations in the time domain and the time lapses were used to calculate the IFs of hemodynamic (i.e., CBF) oscillations and neuronal (i.e., LFP) activities. Our results showed that the CBF mostly oscillated at ~0.1Hz with a full-half-bandwidth of [0.08Hz, 0.15Hz]. In addition, the maximal frequency of LFP firings was also approximately at 0.1Hz, which collaborated with to the frequency of CBF oscillations. Interestingly, CBF increased linearly with the LFP activity up to 0.15Hz (r=0.93), and both signals then decreased rapidly as a function of activity frequency. This indicates the spontaneous hemodynamic LFOs were associated with neuronal activities, thus confirming the neuronal origin of the hemodynamic oscillations.

Transcranial direct current stimulation (tDCS) is a non-invasive cortical stimulation technique that can facilitate task specific plasticity that can improve motor performance. Current tDCS interventions uniformly apply a chosen electrode montage to a subject population without personalizing electrode placement for optimal motor gains. We propose a novel perturbation tDCS (ptDCS) paradigm for determining a personalized electrode montage in which tDCS intervention yields maximal motor performance improvements during stimulation. PtDCS was applied to ten healthy adults and five stroke patients with upper hemiparesis as they performed an isometric wrist flexion task with their non-dominant arm. Simultaneous recordings of torque applied to a stationary handle, muscle activity by electromyography (EMG), and cortical activity by functional near-infrared spectroscopy (fNIRS) during ptDCS helped interpret how cortical activity perturbations by any given electrode montage related to changes in muscle activity and task performance quantified by a Reaction Time (RT) X Error product. PtDCS enabled quantifying the effect on task performance of 20 different electrode pair montages placed over the sensorimotor cortex. Interestingly, the electrode montage maximizing performance in all healthy adults did not match any of the ones being explored in current literature as a means of improving the motor performance of stroke patients. Furthermore, the optimal montage was found to be different in each stroke patient and the resulting motor gains were very significant during stimulation. This study supports the notion that task-specific ptDCS optimization can lend itself to personalizing the rehabilitation of patients with brain injury.

Abundant study on the hemodynamic response of a brain have brought quite a few advances in technologies of measuring it. The most benefitted is the functional near infrared spectroscope (fNIRS). A variety of devices have been developed for different applications. Because portable fNIRS systems were more competent to measure responses either of special subjects or in natural environment, several kinds of portable fNIRS systems have been reported. However, they all required a computer for receiving data. The extra computer increases the cost of a fNIRS system. What’s more noticeable is the space required to locate the computer even for a portable system. It will discount the portability of the fNIRS system. So we designed a self-contained eight channel fNIRS system, which does not demand a computer to receive data and display data in a monitor. Instead, the system is centered by an ARM core CPU, which takes charge in organizing data and saving data, and then displays data on a touch screen. The system has also been validated by experiments on phantoms and on subjects in tasks.

The incidence of perinatal hemorrhagic stroke (HS) is very similar to that in the elderly and produces a significant morbidity and long-term neurologic and cognitive deficits. There is strong evidence that cerebral blood flow (CBF) abnormalities make considerable contribution to HS development. However, the mechanisms responsible for pathological changes in CBF in infants with HS are not established. Therefore, quantitative assessment of CBF may significantly advance the understanding of the nature of neonatal stroke. The aim of this investigation was to determine the particularities of alterations in macro- microcirculation in the brain of newborn rats in the different stages of stress-related development of HS using three-dimensional Doppler optical coherence tomography (DOCT) and laser speckle contrast imaging (LSCI).Our results show that cerebral veins are more sensitive to harmful effect of stress compared with microcirculatory vessels. Stress-induced progressive dilation of cerebral veins with the fall of blood flow velocity precedes HS while pathological changes in microcirculatory vessels are accompanied by development of HS. The further detailed study of cerebral venous and microcirculatory circulation would be a significant advance in development of prognostic criteria for a HS risk during the first days after birthday.

In this series of animal experiments on resuscitation after cardiac arrest we had a unique opportunity to measure hyperspectral near-infrared spectroscopy (hNIRS) parameters directly on the brain dura, or on the brain through the intact pig skull, and simultaneously the muscle hNIRS parameters. Simultaneously the arterial blood pressure and carotid and femoral blood flow were recorded in real time using invasive sensors. We used a novel hyperspectral signalprocessing algorithm to extract time-dependent concentrations of water, hemoglobin, and redox state of cytochrome c oxidase during cardiac arrest and resuscitation. In addition in order to assess the validity of the non-invasive brain measurements the obtained results from the open brain was compared to the results acquired through the skull. The comparison of hNIRS data acquired on brain surface and through the adult pig skull shows that in both cases the hemoglobin and the redox state cytochrome c oxidase changed in similar ways in similar situations and in agreement with blood pressure and flow changes. The comparison of simultaneously measured brain and muscle changes showed expected differences. Overall the results show feasibility of transcranial hNIRS measurements cerebral parameters including the redox state of cytochrome oxidase in human cardiac arrest patients.

Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

Nowadays, there are several imaging techniques offering a complementary approach to visualize intact neural networks on large areas. Each of those offers a different strategy and furnish complementary information on the role of neural components. We will describe different approaches enabling to move from single neuron details to whole brain imaging, connecting short range structural information to long range one. In particular, some examples of correlative microscopies, combining linear and non linear techniques will also be described.

Near-infrared spectroscopy (NIRS) based techniques are utilised in quantifying changes of chromophore concentrations in tissue. Particularly, non-invasive in vivo measurements of tissue oxygenation in the cerebral cortex are of interest. The measurement method is based on illuminating tissue and measuring the back-scattered light at wavelengths of interest. Tissue illumination can be realised using different techniques and various light sources. Commonly, lasers and laser diodes (LD) are utilised, but also high-power light emitting diodes (HPLED) are becoming more common. At the moment, a wide range of available narrow-band light sources exists, covering basically the entire spectrum of interest in brain tissue NIRS measurements. In this paper, in the centre of our interest are LDs and HPLEDs, because of their affordability, efficiency in terms of radiant flux versus size and easiness to adopt in in vivo medical applications. We compare characteristics of LDs and HPLEDs at specific wavelengths and their suitability for in vivo quantifying of different tissue chromophore concentration, particularly in cerebral blood flow (CBF). A special focus is on shape and width of the wavelength bands of interest, generated by the LDs and HPLEDs. Moreover, we experimentally study such effects as, spectroscopy cross talk, separability and signal-to-noise ratio (SNR) when quantifying tissue chromophore concentration. Chromophores of our interest are cytochrome, haemoglobin and water. Various LDs and HPLEDs, producing narrow-band wavelengths in the range from 500 nm to 1000 nm are tested.

Two-layered slab is a rational simplified sample to the near-infrared functional brain imaging using diffuse optical tomography (DOT).The quality of reconstructed images is substantially affected by the accuracy of the background optical properties. In this paper, region step wise reconstruction method is proposed for reconstructing the background optical properties of the two-layered slab sample with the known geometric information based on continuous wave (CW) DOT. The optical properties of the top and bottom layers are respectively reconstructed utilizing the different source-detector-separation groups according to the depth of maximum brain sensitivity of the source-detector-separation. We demonstrate the feasibility of the proposed method and investigate the application range of the source-detector-separation groups by the numerical simulations. The numerical simulation results indicate the proposed method can effectively reconstruct the background optical properties of two-layered slab sample. The relative reconstruction errors are less than 10% when the thickness of the top layer is approximate 10mm. The reconstruction of target caused by brain activation is investigated with the reconstructed optical properties as well. The quantitativeness ratio of the ROI is about 80% which is higher than that of the conventional method. The spatial resolution of the reconstructions (R) with two targets is investigated, and it demonstrates R with the proposed method is better than that with the conventional method as well.

Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer’s disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca²⁺ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca²⁺ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca²⁺ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson’s disease in a preclinical study.

Transvascular drug delivery to the brain is difficult due to the blood-brain barrier (BBB). Thus, various methods for safely opening the BBB have been investigated, for which real-time imaging methods are desired both for the blood vessels and distribution of a drug. Photoacoustic (PA) imaging, which enables depth-resolved visualization of chromophores in tissue, would be useful for this purpose. In this study, we performed in vivo PA imaging of the blood vessels and distribution of a drug in the rat brain by using an originally developed compact PA imaging system with fiber-based illumination. As a test drug, Evans blue (EB) was injected to the tail vein, and a photomechanical wave was applied to the targeted brain tissue to increase the permeability of the blood vessel walls. For PA imaging of blood vessels and EB distribution, nanosecond pulses at 532 nm and 670 nm were used, respectively. We clearly visualized blood vessels with diameters larger than 50 μm and the distribution of EB in the brain, showing spatiotemporal characteristics of EB that was transvascularly delivered to the target tissue in the brain.

Due to considerable increase in the terrorism using explosive devices, blast-induced traumatic brain injury (bTBI) receives much attention worldwide. However, little is known about the pathology and mechanism of bTBI. In our previous study, we found that cortical spreading depolarization (CSD) occurred in the hemisphere exposed to a laser- induced shock wave (LISW), which was followed by long-lasting hypoxemia-oligemia. However, there is no information on the events occurred in the contralateral hemisphere. In this study, we performed multichannel fiber-based diffuse reflectance spectroscopy for the rat brain exposed to an LISW and compared the results for the ipsilateral and contralateral hemispheres. A pair of optical fibers was put on the both exposed right and left parietal bone; white light was delivered to the brain through source fibers and diffuse reflectance signals were collected with detection fibers for both hemispheres. An LISW was applied to the left (ipsilateral) hemisphere. By analyzing reflectance signals, we evaluated occurrence of CSD, blood volume and oxygen saturation for both hemispheres. In the ipsilateral hemispheres, we observed the occurrence of CSD and long-lasting hypoxemia-oligemia in all rats examined (n=8), as observed in our previous study. In the contralateral hemisphere, on the other hand, no occurrence of CSD was observed, but we observed oligemia in 7 of 8 rats and hypoxemia in 1 of 8 rats, suggesting a mechanism to cause hypoxemia or oligemia or both that is (are) not directly associated with CSD in the contralateral hemisphere.

The development of various optical clearing methods provides a great potential for imaging entire central nervous system by combining with multiple-labelling and microscopic imaging techniques. These methods had made certain clearing contributions with respective weaknesses, including tissue deformation, fluorescence quenching, execution complexity and antibody penetration limitation that makes immunostaining of tissue blocks difficult. The passive clarity technique (PACT) bypasses those problems and clears the samples with simple implementation, excellent transparency with fine fluorescence retention, but the passive tissue clearing method needs too long time. In this study, we not only accelerate the clearing speed of brain blocks but also preserve GFP fluorescence well by screening an optimal clearing temperature. The selection of proper temperature will make PACT more applicable, which evidently broaden the application range of this method.

We report on a novel miniature head-mounted imaging system for simultaneous optical recording of brain blood flow and changes in brain blood oxygenation in a rat. Measurements of blood flow speeds are accomplished using Laser Speckle Contrast Imaging (LSCI) technique, while changes in blood oxygenation are measured via Intrinsic Optical Signal Imaging (IOSI) technique. A single multi-wavelength (wavelength = 680, 795, 850 nm) package of vertical cavity surface emitting lasers (VCSELs) is used as the sole brain illumination source. VCSELs enable rapid toggling between wavelengths, and between high-coherence and low-coherence modes, necessary for LSCI and IOSI, respectively. The combination of a miniature light source and a small 10-bit CCD camera sensor lead to a sub-20 g device mass. The miniature imaging system, including the lens, camera, and illumination lasers, is packaged as a module, and is mounted on a chronic implanted observation window that is surgically placed in the skull, allowing for repeated measurements and removal of the imaging system from the rats head after the imaging session. The imaging system allows for a 2mm-diameter field of view and a resolution of 7.4 µm. It will allow neurophysiologists to correlate standard behavioural assays to neurovascular response in animal models, and thus enrich their understanding of neurovascular coupling dynamics of brain disorders and diseases such as stroke and epilepsy.

Driver fatigue is one of the leading causes of traffic accidents. It is imperative to develop a technique to monitor fatigue of drivers in real situation. Near-infrared spectroscopy (fNIRS) is now capable of measuring brain functional activity noninvasively in terms of hemodynamic responses sensitively, which shed a light to us that it may be possible to detect fatigue-specified brain functional activity signal. We developed a sensitive, portable and absolute-measure fNIRS, and utilized it to monitor cerebral hemodynamics on car drivers during prolonged true driving. An odd-ball protocol was employed to trigger the drivers’ visual divided attention, which is a critical function in safe driving. We found that oxyhemoglobin concentration and blood volume in prefrontal lobe dramatically increased with driving duration (stand for fatigue degree; 2-10 hours), while deoxyhemoglobin concentration increased to the top at 4 hours then decreased slowly. The behavior performance showed clear decrement only after 6 hours. Our study showed the strong potential of fNIRS combined with divided visual attention protocol in driving fatigue degree monitoring. Our findings indicated the fNIRS-measured hemodynamic parameters were more sensitive than behavior performance evaluation.

Although several studies have shown the feasibility of using optogenetics in non-human primates (NHP), reliable largescale chronic interfaces have not yet been reported for such studies in NHP. Here we introduce a chronic setup that permits repeated, daily optogenetic stimulation and large-scale recording from the same sites in NHP cortex. The setup combines optogenetics with a transparent artificial dura (AD) and high-density micro-electrocorticography (μECoG). To obtain expression across large areas of cortex, we infused AAV5-CamKIIa-C1V1-EYFP viral vector using an infusion technique based on convection-enhanced delivery (CED) in primary somatosensory (S1) and motor (M1) cortices. By epifluorescent imaging through AD we were able to confirm high levels of expression covering about 110 mm² of S1 and M1. We then incorporated a 192-channel μECoG array spanning 192 mm² into the AD for simultaneous electrophysiological recording during optical stimulation. The array consists of patterned Pt-Au-Pt metal traces embedded in ~10 μm Parylene-C insulator. The parylene is sufficiently transparent to allow minimally attenuated optical access for optogenetic stimulation. The array was chronically implanted over the opsin-expressing areas in M1 and S1 for over two weeks. Optical stimulation was delivered via a fiber optic placed on the surface of the AD. With this setup, we recorded reliable evoked activity following light stimulation at several locations. Similar responses were recorded across tens of days, however a decline in the light-evoked signal amplitude was observed during this period due to the growth of dural tissue over the array. These results show the feasibility of a chronic interface for combined largescale optogenetic stimulation and cortical recordings across days.

In this paper we propose using Array Waveguide Gratings (AWGs), working in the visible range, in order to implement the technique of Wavelength-Division-(de)Multiplexing for multi-point stimulation of deep-brain neurons. We've developed a CMOS compatible fabrication process and fabricated two sets of AWGs, working in the red and blue wavelengths. Experimental data demonstrating the functionality of these AWGs is presented.

Molecular dynamics at synaptic terminals in neuronal cells is essential for synaptic plasticity and subsequent modulation of cellular functions in a neuronal network. For realizing artificial control of living neuronal network, we demonstrate laser-induced perturbation into molecular dynamics in the neuronal cells. The optical trapping of cellular molecules such as synaptic vesicles or neural cell adhesion molecules labeled with quantum dots was evaluated by fluorescence imaging and fluorescence correlation spectroscopy. The trapping and assembling dynamics was revealed that the molecular motion was constrained at the focal spot of a focused laser beam due to optical trapping force. Our method has a potential to manipulate synaptic transmission at single synapse level.

Multipoint Light Emitting Optical Fibers (MPF) has been recently demonstrated as a versatile tool for spatially addressable optogenetics experiments. Their fabrication has been possible thanks to a number of key microfabrication technologies, in particular the unique nanofabrication capabilities of a Focused Ion Beam. This work provides the complete description of MPF fabrication, detailing the optimization process for each fabrication step.

Non-invasive remote control technologies designed to manipulate neural functions for a comprehensive and quantitative understanding of the neuronal network in the brain as well as for the therapy of neurological disorders have long been awaited. Recently, it has become possible to optically manipulate the neuronal activity using biological photo-reactive molecules such as channelrhodopsin-2 (ChR2). However, ChR2 and its relatives are mostly reactive to visible light which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light penetrates deep into the tissues because biological systems are almost transparent to light within this so-called ‘imaging window’. Here we used lanthanide nanoparticles (LNPs), which are composed of rare-earth elements, as luminous bodies to activate channelrhodopsins (ChRs) since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Neuron-glioma-hybrid ND-7/23 cells were cultured with LNP(NaYF₄:Sc/Yb/Er) particles (peak emission, 543 nm) and transfected to express C1V1 (peak absorbance, 539 nm), a chimera of ChR1 and VChR1. The photocurrents were generated in response to NIR laser light (976 nm) to a level comparable to that evoked by a filtered Hg lamp (530-550 nm). NIR light pulses also evoked action potentials in the cultured neurons that expressed C1V1. It is suggested that the green luminescent light emitted from LNPs effectively activated C1V1 to generate the photocurrent. With the optimization of LNPs, acceptor photo-reactive biomolecules and optics, this system could be applied to non-invasively actuate neurons deep in the brain.

The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called “optogenetic” technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

Micromirror arrays (MMA) are spatial light modulators (SLM) used in a wide variety of applications for structured light manipulation i.e. structured illumination microscopy.

In our setup, we use a combination of two micromirror arrays, which allow not only to spatially structure the light in the field of view, but also to control the direction and angle of the incident light. In order to achieve this, a first MMA is imaged in the focal plane and used as a black and white (or even greyscale) mask. With a fully illuminated objective, this image would normally be formed from the complete light cone. By imaging the second MMA onto the backfocal plane of the objective only a portion of the light cone is used to form the image. This enables avoiding the unwanted illumination of out of focus objects. The MMAs in our setup consist of an array of 256x256 micromirrors, that can each be individually and continuously tilted up to 450nm, allowing the creation of greyscale images in real time in the illumination pattern. The mirrors themselves can be tilted for times as short as 10μs up to several seconds. This gives unprecedented control over the illumination times and intensities in the sample. Furthermore, our enhanced coating technology yields a high reflectivity over a broad optical spectrum (240- 1000nm).

Overall, the setup allows targetted illumination of subcellular regions enabling the precise, localized activation of optogenetic probes or the activation and deactivation of signaling cascades using photo-activated ion-channels.

Fluorescent Light-inducible Proteins are proteins of interest that have Dronpa domains fused to both termini. In the dark, the Dronpa domains tetramerize and cage the protein, but light induces Dronpa dissociation and activates the protein. Here, we describe four experimental systems for light activation of mammalian cells expressing FLiPs.

Many people suffering from diseases such as epilepsy, autism, or are diagnosed with schizophrenia, Alzheimer's, or Parkinson's disease that cannot be helped because we do not understand how the brain works, notes Rafael Yuste in this BiOS Hot Topic talk. So scientists are working on methodologies to study the brain, visualize neurons, and map the connections, in order to comprehend neural circuitry in its entirety and develop treatments. Neuroscientists are currently able to visualize neuron activity using calcium imaging, based on changes in fluorescence intensity or spectral properties of a dye that is sensitive to fluctuations in intracellular calcium concentrations, which is directly related with neuron activity. This method works in live animals, using a window in their skull. Nonlinear microscopy provides the ability to image deeper inside the brain. The Yuste group is now working on expanding this technique to fast 3D imaging in the brains of living animals. Until not long ago, 3D visualization was only achievable by sequentially scanning different focal planes, which is very time-consuming. The implementation of holographic methods in their microscope can bring a solution. Rafael Yuste is Professor of Biological Sciences and Neuroscience at Columbia University. He obtained his MD at the Universidad Autónoma in the Fundación Jimenez Diaz Hospital (Spain). After a brief research period in Sydney Brenner's group at the LMB in Cambridge, UK, he performed PhD studies with Larry Katz in Torsten Wiesel's laboratory at Rockefeller University in New York. He then moved to Bell Labs, where he was a postdoctoral student of David Tank and Winfried Denk. In 2005 he became HHMI Investigator and Co-Director of the Kavli Institute for Brain Science at Columbia. He has been a visiting researcher in Javier DeFelipe's laboratory at the Cajal Institute/UPM in Madrid since 1997, and since 2012 at the Allen Institute for Brain Science in Seattle.

In this Hot Topics presentation, David Roberts describes recent work in image-guided neurosurgery. Gliomas can present a challenge for surgical removal because they invade normal brain tissue that may be highly functional, so it is crucial to develop techniques for improved visualization of the tumor's margins. Surgical removal of a brain tumor can be aided using a technique in which tumor tissue is fluoresced during surgery. Roberts received an A.B. degree from Princeton University, an M.A. degree from Oxford University, and an M.D. degree from Dartmouth Medical School. He interned in surgery at the University of Utah and did his neurosurgical residency at Dartmouth. In 1982, he jointed the staff of the Hitchcock Clinic and the faculty of Dartmouth Medical School, at the rank of Assistant Professor. He was promoted to Associate Professor in 1988 and Professor in 1994. In 1997 he became Chair of the Section of Neurosurgery at the Dartmouth-Hitchcock Medical Center and Director of the neurosurgical residency training program. While retaining those responsibilities, he was appointed in 2000 to the position at Dartmouth Medical School of Senior Associate Dean for Clinical Affairs, and the following year to the Alma Hass Milham Distinguished Chair in Clinical Medicine. His present research interests are in epilepsy, stereotaxy and computer-assisted surgery. He has been the author or co-author of more than 100 publications and has co-edited six books and monographs.