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- Front Matter: Volume 8928
- Microscopy and OCT I
- Microscopy and OCT II
- Operative and Post-op Therapy I
- Operative and Post-op Therapy II
- Optical Spectroscopy and Tomography: Pre-Clinical
- Optical Spectroscopy and Tomography: Clinical
- Poster Session
- Novel Photonic/Optoelectronic Methods and Applications II
- Optical Neuroimaging I
- Visible Brainwide Networks II
- Optical Neuroimaging IV
- Poster Session
- Optogenetics II
- Optical Control of Cells II
- Optogenetics III
- Hot Topics Session
Front Matter: Volume 8928
Front Matter: Volume 8928
Show abstract
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.
Microscopy and OCT I
Optical coherence microscopy of mouse cortical vasculature surrounding implanted electrodes
Show abstract
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.
Microscopy and OCT II
Interpreting CARS images of tissue within the C-H-stretching region
Show abstract
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.
Raman spectroscopy of gliomas: an exploratory study
Mahesh Shenoy,
Arti R. Hole,
E. Shridhar,
et al.
Show abstract
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.
Operative and Post-op Therapy I
Quantitative spectrally resolved intraoperative fluorescence imaging for neurosurgical guidance in brain tumor surgery: pre-clinical and clinical results
Show abstract
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.
Photochemical internalization (PCI) enhanced nonviral transfection of tumor suppressor and pro-drug activating genes; a potential treatment modality for gliomas
Frederick Wang,
Genesis Zamora,
Chung-Ho Sun,
et al.
Show abstract
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).
Mid-IR laser system for advanced neurosurgery
M. Klosner,
C. Wu,
D. F. Heller
Show abstract
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.
Operative and Post-op Therapy II
5-ALA based photodynamic management of glioblastoma
Show abstract
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.
Ultra-low fluence rate photodynamic therapy: simulation of light emitted by the Cerenkov effect
Jonathan Gonzales,
Fred Wang,
Genesis Zamora,
et al.
Show abstract
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.
Optical Spectroscopy and Tomography: Pre-Clinical
Combining ICA and Granger causality: a novel tool for investigation of brain dynamics and brain oscillations using
fNIRS measurements
Show abstract
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.
Monitoring closed head injury induced changes in brain physiology with orthogonal diffuse near-infrared reflectance
spectroscopy
Show abstract
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.
Near-infrared diffuse reflectance imaging of infarct core and peri-infarct depolarization in a rat middle cerebral
artery occlusion model
Show abstract
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.
Implantable CMOS imaging device with absorption filters for green fluorescence imaging
Show abstract
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.
In vivo imaging of scattering and absorption properties of exposed brain using a digital red-green-blue camera
Show abstract
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 μs'=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.
Optical Spectroscopy and Tomography: Clinical
Hemodynamic measurements in deep brain tissues of humans by near-infrared time-resolved spectroscopy
Show abstract
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.
Noninvasive optical evaluation of low frequency oscillations in prefrontal cortex hemodynamics during verbal working memory
Show abstract
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.
Poster Session
Development of a fiber-less fNIRS system and its application to hair-covered head
Show abstract
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.
Precise spatial co-registration in simultaneous fNIRS and fMRI measurements using markers coaxially fixable to the optodes
Show abstract
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.
Technical considerations on confocal based fluorescence micro-optical sectioning tomography for visualizing brain circuits
Show abstract
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.
Photothermal therapy of human glioma spheroids with gold-silica nanoshells and gold nanorods: a comparative study
Show abstract
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.
Activity-dependent signal changes in neurons by fiber-coupled microscopy
Show abstract
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.
Novel Photonic/Optoelectronic Methods and Applications II
Studying hemispheric lateralization during a Stroop task by near-infrared spectroscopy
Show abstract
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.
Quantitative assessment of brain tissue oxygenation in porcine models of cardiac arrest and cardiopulmonary resuscitation using
hyperspectral near-infrared spectroscopy
Show abstract
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.
Optical stimulation of the hearing and deaf cochlea under thermal and stress confinement condition
Show abstract
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) inhibits electrically evoked neural responses in the deaf white cat
Show abstract
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.
Target structures in the cochlea for infrared neural stimulation (INS)
Show abstract
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.
Optical Neuroimaging I
Development of optical neuroimaging to detect drug-induced brain functional changes in vivo
Show abstract
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.
Imaging of rat brain using short graded-index multimode fiber
Show abstract
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.
Visible Brainwide Networks II
In vivo imaging of neural reactive plasticity after laser axotomy in cerebellar cortex
Show abstract
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.
Optical Neuroimaging IV
Astrocytic adaptation during cerebral angiogenesis follows the new vessel formation induced through chronic hypoxia in adult mouse cortex
Kazuto Masamoto,
Iwao Kanno
Show abstract
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.
Poster Session
Cerebral hemodynamics in patients with obstructive sleep apnea syndrome monitored with near-infrared spectroscopy (NIRS) during positive airways pressure (CPAP) therapy: a pilot study
Show abstract
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.
A pilot study to compare the cerebral hemodynamics between patients with obstructive sleep apnea syndrome (OSA) and periodic limb movement syndrome (PLMS) during nocturnal sleep with near-infrared spectroscopy (NIRS)
Show abstract
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.
The effect of RGB monochromatic and polychromatic LED lighting on growth performance, behavior, and development of broilers
Waldirene B. B. Morrill,
Janice M. C. Barnabé,
Tatiana P. N. da Silva,
et al.
Show abstract
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.
Optical monitoring of shock wave-induced spreading depolarization and concomitant hypoxemia in rat brain
Show abstract
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.
Optogenetics II
Non-invasive activation of optogenetic actuators
Elisabeth Birkner,
Ken Berglund,
Marguerita E. Klein,
et al.
Show abstract
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.
Optical Control of Cells II
Fiber optic fluorescence microscopy for functional brain imaging in awake, mobile mice
Show abstract
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.
Optogenetics III
Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro
Show abstract
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.
Hot Topics Session
New small quantum dots for neuroscience (Presentation Video)
Paul Selvin
Show abstract
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.
Journey into the brain: from single synapse to whole brain anatomy by correlative microscopy (Presentation Video)
Francesco Pavone
Show abstract
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.