This PDF file contains the front matter associated with SPIE Proceedings Volume 10493 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

I will present the fundamentals of deep tissue, non-invasive measurement of blood flow using diffuse speckle statistics and provide examples of different technological examples alongside clinical applications. I will conclude with the latest developments in the field towards fast, time-resolved, portable, low-cost systems.

The laser speckle flowmetry methods based on laser speckle imaging (LSI) have attracted extensive attention recently because they can image brain blood flow with high spatiotemporal resolution. However, the poor transparency of the cranial bone limits the spatial resolution and the imaging depth. This problem has previously been addressed in animal studies by removing or thinning the skull to transparency. Nevertheless, a permanent and reliable solution has not yet been developed. Our study demonstrates a new method to address this challenge in biomedical imaging research, through the use of novel transparent cranial implants made from nanocrystalline yttria-stabilized zirconia (nc-YSZ). By applying LSI to underlying brain in an acute murine model, we show that spatial resolution and quantitative accuracy of blood flow measurement are improved when imaging through transparent nc-YSZ implants relative to native cranium. As such, these results provide the initial evidence supporting the feasibility of nc-YSZ transparent cranial implant as a clinically-viable long-term optical access for LSI on a chronically-recurring basis, thereby suppressing the need for repeated craniotomies. Successful development of this method has the potential to advance the study of neuropathologies or novel neuro-procedures in animal models where measurement of cerebral blood flow is of interest, such as blood flow changes during stroke, changes in blood flow due to functional activation, and spreading depolarization and its role in brain injuries, pathophysiology of migraine, and subarachnoid hemorrhage.

Biodynamic imaging uses coherence-gated dynamic light scattering to create three dimensional maps of intracellular dynamics in living tissue biopsies. The technique is sensitive to changes in intracellular dynamics dependent on the mechanism of action (MoA) of therapeutics applied in vitro to the living samples. A preclinical trial in the assessment of chemotherapeutic response of dogs with B-cell lymphoma to the doxorubicin-based therapy CHOP has been completed using biodynamic imaging. The trial enrolled 19 canine patients presenting with non-Hodgkin’s B-cell lymphoma. Biopsies were acquired through surgery or through needle cores. The time-varying power spectrum of scattered light after drugs are applied ex vivo to the biopsies represent biodynamic biomarkers that are used in machine learning algorithms to predict the patient clinical outcome. Two distinct phenotypes emerged from the analysis that correlate with patient drug resistance or sensitivity. Cross validation of the algorithms perform with an accuracy of 90% in the prediction of dogs that will respond to treatment. Biodynamic imaging has the potential to help select chemotherapy for personalized cancer care.

Light scattered from dynamic random media has numerous applications in medicine, biology, engineering, physics and numerous other fields. Short term and long term variations and correlations in the scattered intensity (“speckle”) provide information about the crossing of scattering paths as a result of the local structure and dynamics within the medium. Poincaré descriptors are statistical tools used to study variations or self-similarity in neighboring values of a quantity. Herein, we modify this definition to examine correlations not only between neighboring (temporally and spatially) values of dynamic speckle patterns, but also between values with larger spatial and temporal distances between them. The effects of incoherently summed, that is, time-averaged speckle patterns will be examined, as will be the separate cases of incoherently summed correlated and un-correlated speckle patterns. The unique case of elongated speckle will also be presented. The ratio of short-term to long-term differences in the pattern, a term referred to as the ‘ellipticity’ of the data, yields information on the dominance of long-term variations in the scattered intensity compared to the short-term variations. We will show that Poincaré descriptors are useful in quantifying the width of the coherence areas in all 3 dimensions in the scattered intensity patterns and also for quantifying motions in speckle patterns from which information about the dynamics of the medium can be inferred.

In smart minimally invasive devices, signal transmission and supply of power to the distal end require many thin and fragile wires in order to keep the catheter slim and flexible. We replaced electrical wires by optical fibers and show signal transfer of synthetic aperture ultrasound images as well as photo-voltaic conversion to supply all electronics The absence of conductors provides both intrinsic galvanic isolation and magnetic resonance imaging (MRI) compatibility. The simple and cost-effective design may be pivotal for translation of these advanced devices into the clinic.

Blood flow, heart contraction, and tissue stiffness are important regulators of cardiac morphogenesis and function during embryonic development. Defining how these factors are integrated is critically important to advance prevention, diagnostics, and treatment of congenital heart defects. Mammalian embryonic development is taking place deep within the female body, which makes cardiodynamic imaging and analysis during early developmental stages in humans inaccessible. With thousands of mutant lines available and well-established genetic manipulation tools, mouse is a great model to understand how biomechanical factors are integrated with molecular pathways to regulate cardiac function and development. Dynamic imaging and quantitative analysis of the biomechanics of live mouse embryos have become increasingly important, which demands continuous advancements in imaging techniques and live assessment approaches. This has been one of the major drives to keep pushing the frontier of embryonic imaging for better resolution, higher speed, deeper penetration, and more diverse and effective contrasts. Optical coherence tomography (OCT) has played a significant role in addressing such demands, and its features in non-labeling imaging, 3D capability, a large working distance, and various functional derivatives allow OCT to cover a number of specific applications in embryonic imaging. Recently, our group has made several technical improvements in using OCT to probe the biomechanical aspects of live developing mouse embryos at early stages. These include the direct volumetric structural and functional imaging of the cardiodynamics, four-dimensional quantitative Doppler imaging and analysis of the cardiac blood flow, and fourdimensional blood flow separation from the cardiac wall tissue in the beating embryonic heart. Here, we present a short review of these studies together with brief descriptions of the previous work that demonstrate OCT as a valuable and useful imaging tool for the research in developmental cardiology.

Background: Examination of the skin’s vascular and structural features is essential in clinical, medical, and research dermatology. However, there is a lack of comprehensive imaging tools that clearly and accurately evaluates the skin’s vascular and structural features. Current techniques are invasive and have inherent preparatory drawbacks. Aim: To use optical coherence tomography angiography (OCTA) for a more accurate depiction of vessels in the skin without the use of a dye or other invasive techniques to visualize and asses the role of the skin’s vasculature during the process’ of wound healing. Method: We used an in-house-built, swept source-OCT system to perform OCTA analyses so as to image the vascular features of a cutaneous wound to a depth of 1.2 mm as it was healing. Key vascular parameters, such as vessel density and diameter, were measured at various depths to elucidate how depth might influence the vascular response. Observation: We found that alterations to the vasculature of the skin are linked to active healing. The first response of the superficial vessels observed here is to increase in diameter, whilst the first response of the deeper vessels is to increase in density. Additionally, the superficial vessels appear to normalize at an earlier compared to deeper vessels. Conclusion: OCTA is capable of imaging and distinguishing the complex collection of events that play pivotal roles in the repair of healthy skin that could be useful in the assessment of skin repair and treatment after injury or surgery.

Swept-source optical coherence tomography (SS-OCT) is gradually out-performing spectral-domain OCT (SD-OCT) in many aspects including sensitivity, speed, and ranging distance. However, its phase-stability is generally more difficult to achieve, compared to SD-OCT, which limited the functional imaging applications of phase-sensitive SSOCT. In this study, a novel phase stabilization technique is demonstrated with significant improvement in the phase stability of an SS-OCT system that is based on micro-electromechanical (MEMS) vertical cavity surface-emitting laser (VCSEL). Without any requirements of hardware modifications, this numerical phase stabilization technique features high tolerance to acquisition jitter, and significantly reduced budget in computational effort. We demonstrate that when measured with biological tissue, this technique enables a phase sensitivity of 89 mrad in highly scattering tissue, with image ranging distance of up to 12.5 mm at A-line scan rate of 100.3 kHz. We further compare the performances delivered by the phase-stabilization approach with conventional numerical approach for accuracy and computational efficiency. Imaging result of complex signal-based optical coherence tomography angiography (OCTA) and Doppler OCTA indicate that the proposed phase stabilization technique is robust, and efficient in improving the image contrast-to-noise ratio and extending OCTA depth range. The proposed technique can be universally applied to improve phase-stability in generic SS-OCT with different scale of scan rates without special hardware or extra imaging operations.

Penetrating vessels bridge the mesh of communicating vessels on the surface of cortex with the subsurface microvascular bed that feeds the underlying neural tissue. The degeneration and dysfunction of penetrating vessels directly relates to Alzheimer’s disease, perceptual deficit, amnestic syndrome and stroke. Here we propose a cerebral penetrating vessel mapping approach based on eigen decompensation (ED) principle component analysis that is innovatively redesigned from optical coherence tomography (OCT) angiography. Ensemble complex OCT signals acquired through repeated A-scans first form a covariance matrix and then project into an eigenspace to represent frequency components of moving particles. The eigen representation of signals possesses several advantages over that in spatiotemporal domain: 1) the eigen components possess distinct statistical distributions for penetrating vessels, surface communicating vessels, vessel free regions, and territories occupied by enriched capillaries; 2) this approach is immune to tailing artifacts, enabling automatic decoupling of penetrating vessels from lateral vasculature networks. To describe the uniqueness of penetrating vessels as 2D parameter mapping, a second round of eigen analysis is applied to the eigen representations by taking each eigen component as an observation and distributions of the eigen components as features. In our datasets of mouse cerebral cortex, the eigen components mainly follow a subtle logistic distribution, statistically more significant than other features in terms of distribution spectral power (> 30 dB). While, the existence of vessel penetrating behavior locally breaks this distribution, assigning low transform probabilities to corresponding A-scans. Therefore, the transform coefficients inversely correlate to the vessel penetration and fully reveal the spatial morphology of penetrating vessels from projection view. This method allows for automatic statistical quantification of penetrating arterioles and ascending venules from large volume OCT angiography data, and accordingly contributes to the morphometric analysis of cortical microvasculature in functioning brains.

Optical coherence angiography (OCA) enables visualisation of three-dimensional micro-vasculature from optical coherence tomography data volumes. Typically, various statistical methods are used to discriminate static tissue from blood flow within vessels. In this paper, we introduce a new method that relies upon the beating heart frequency to isolate blood vessels from the surrounding tissue. Vascular blood flow is assumed to be more strongly modulated by the heart-beat compared to surrounding tissue and therefore short-time Fourier transform of sequential measurements can discriminate the two. Furthermore, it is demonstrated that adjacent B-Scans within an OCT data volume can provide the required sampling frequency. As such, the technique can be considered to be a spatially mapped variation of photoplethysmography (PPG), whereby each image voxel operates as a PPG detector. This principle is demonstrated using both a model system and in vivo for monitoring the vascular changes effected by traumatic brain injury in mice. In vivo measurements were acquired at an A-Scan rate of 10kHz to form a 500x500x512 (lateral x lateral x axial) pixel volume, enabling sequential sampling of the mouse heart rate in an expected range of 300-600 bpm. One of the advantages of this new OCA processing method is that it can be used in conjunction with existing algorithms as an additional filter for signal to noise enhancement.

Cerebral microvascular changes are influenced by intracranial pressure (ICP) as well as mean arterial blood pressure (MAP). The mechanism maintaining blood flow despite changes in either pressure is called cerebral autoregulation. This mechanism is known to be impaired in many diseases, including traumatic brain injury and stroke. Maintaining adequate cerebral blood flow and autoregulation is known to improve long term patient outcomes. However, the influence on the microvasculature and autoregulation of blood pressure vs. fluid increase, hence intracranial pressure, is not well understood. Furthermore, while blood pressure changes can readily be measured, intracranial pressure sensors are invasive and there is a need to overcome this invasiveness. We have recently shown that changes in cerebral perfusion pressure, which is the difference between blood pressure and intracranial pressure, can be correlated to total hemoglobin concentration, as measured non-invasively with near-infrared spectroscopy (NIRS) in non-human primates. These results showed that non-invasive intracranial pressure monitoring should be possible by means of vascular changes as measured with NIRS. In order to quantify autoregulation and differentiate between blood pressure and fluid increase driven vascular changes, we collected data on non-human primates. The primates’ brains were cannulated to induce rapid changes in ICP. Exsanguination was performed to reduce blood pressure. Data was collected with a combined frequency domain NIRS (OxiplexTS, ISS Inc.) and diffuse correlation spectroscopy (DCS) system for measuring hemoglobin concentration changes as well as blood flow changes, respectively. We will present on the experimental implementation as well as data analysis for quantifying cerebral autoregulation.

The adult zebrafish has pronounced regenerative capacity of the brain, which makes it an ideal model organism of vertebrate biology for the investigation of recovery of central nervous system injuries. The aim of this study was to employ spectral-domain optical coherence tomography (SD-OCT) system for long-term in vivo monitoring of tissue regeneration using an adult zebrafish model of brain injury. Based on a 1325 nm light source and two high-speed galvo mirrors, the SD-OCT system can offer a large field of view of the three-dimensional (3D) brain structures with high imaging resolution (12 μm axial and 13 μm lateral) at video rate. In vivo experiments based on this system were conducted to monitor the regeneration process of zebrafish brain after injury during a period of 43 days. To monitor and detect the process of tissue regeneration, we performed 3D in vivo imaging in a zebrafish model of adult brain injury during a period of 43 days. The coronal and sagittal views of the injured zebrafish brain at each time point (0 days, 10 days, 20 days and 43 days postlesion) were presented to show the changes of the brain lesion in detail. In addition, the 3D SD-OCT images for an injured zebrafish brain were also reconstructed at days 0 and days 43 post-lesion. We found that SD-OCT is able to effectively and noninvasively monitor the regeneration of the adult zebrafish brain after injury in real time with high 3D spatial resolution and good penetration depth. Our findings also suggested that the adult zebrafish has the extraordinary capability of brain regeneration and is able to repair itself after brain injury.

Normal aging is associated with various metabolic and vascular changes. In the brain, the aging leads to an impairment of vessel structure and function. Characterizing the cerebrovascular pathologies with age is of importance to elucidate the underlying mechanism of cognitive decline correlated with blood perfusion. Here, we examine the effect of aging on cerebral microcirculation up to a capillary flow scale. This study uses optical coherence tomography angiography (OCTA) to measure vessel tortuosity, red blood cell (RBC) speed in individual capillaries and capillary density in the sensory-motor cortex of 8 young (3-month-old) and 8 aged (16-month-old) mice under isoflurane anesthesia. The result shows that the surface arterial vessels are more tortuous and the capillary RBC speed is much higher in aged animals old compared with young ones. However, the capillary vessel density is significantly lowered in the aged group than the young group.

The cerebral vascular system serves constant demand of neuronal activities in the brain. Neural activations are typically followed by immediate rise in local blood flow through neural-vascular coupling. Temporal dynamics and spatial redistribution of this hyperemia within the capillary bed play a deterministic role in oxygen diffusing capacity, however, the functional behavior of which remains poorly understood. Taking the advantages of the high spatiotemporal resolution of OCT velocimetry designed upon eigen-decomposition (ED) statistical analysis, we investigated the intrinsic capillary red blood cell (RBC) fluctuations within mouse cerebral cortex, representing as bandwidths of the RBC flow frequencies. The temporal hemodynamics before (rest) and during (activation) a bout of hindpaw electrical stimulations are accordingly analyzed to resolve alterations in capillary flow disturbance and its spatial distribution. In our experiment, the electrical stimulation provokes a temporal RBC fluctuation increase (rest: 16715 m/s; activation: 20516 m/s; P < 0.05) in the capillary bed located in hindpaw somatosensory cortex (HSC), as compared to the control (rest: 17020 m/s; activation: 16918 m/s; P > 0.05) ; accompanied with an increase in capillary RBC velocity (rest: 49640 m/s; activation: 61349 m/s; P < 0.05) in HSC, as compared to the control (rest: 54462 m/s; activation: 55868 m/s; P > 0.05). In addition, no significant difference was observed for the capillary vessel density in either HSC (rest: 0.390.02 m/s; activation: 0.370.01 m/s; P > 0.05) or control (rest: 0.360.02 m/s; activation: 0.370.02 m/s; P > 0.05). We conclude that neural activation evokes spatiotemporal redistribution of capillary hemodynamics regulated through instantaneous increments in flow disturbance and flow velocity, but involves no recruitment of reserved capillaries (no RBC transit path variation). Our demonstration shows the potential of OCT angiography for functional investigation and modeling of spatiotemporally coupled hemodynamics to neural activities.

Although chemotherapeutics for cancer treatment are becoming increasingly efficient these days, they often cause severe dermal side effects. Systemically applied doxorubicin is known for inducing free radicals, which leads to the development of the hand-foot syndrome. This syndrome manifests itself through skin irritations, extending from blistering to open wounds. As doxorubicin exhibits a fluorescence signal in the 520-600 nm region if excited at 488 nm, the doxorubicin’s leakage onto the skin surface could be analyzed. It was found that part of the doxorubicin is ejected with the sweat onto the skin surface, where it spreads and penetrates into the skin like topically applied. By topical application of antioxidants, the doxorubicin could be prevented from inducing free radicals in the skin and consequently the hand-foot syndrome. Raman spectroscopy was used to show that the action mechanism of chemotherapeutics not showing fluorescence signals is similar to the action mechanism of doxorubicin.

Hypoxia, or low oxygen partial pressure, is a common feature of many solid tumours, associated with therapy resistance and poor prognosis for cancer patients. This chemical feature of the tumour microenvironment represents an imbalance of blood oxygen supply and tissue oxygen demand. Given the prognostic significance of hypoxia, there is a clinical unmet need to provide validated, non-invasive, imaging biomarkers that can be used to detect and monitor the spatiotemporal distribution of hypoxia at the point of cancer diagnosis and during treatment. In this talk, I will give an overview of how photoacoustic imaging can address this unmet need, providing examples from both preclinical studies in mouse models of cancer and clinical studies in human patients.

Polarization imaging is regarded as a promising technique for probing the microstructures, especially the anisotropic fibrous components of tissues. Among the available polarimetric techniques, Mueller matrix imaging has many distinctive advantages. Recently, we have developed a Mueller matrix microscope by adding the polarization state generator and analyzer to a commercial transmission-light microscope, and applied it to differentiate human liver and cervical cancerous tissues with fibrosis. Here we apply the Mueller matrix microscope for quantitative detection of human breast ductal carcinoma, which is a primary form of breast cancers, at different stages. The Mueller matrix polar decomposition (MMPD) and Mueller matrix transformation (MMT) parameters of the breast ductal tissues in different regions at in situ and invasive stages are calculated and analyzed. For more comparisons, Monte Carlo simulations based on the sphere-birefringence model are also carried out. The experimental and simulated results indicate that the Mueller matrix microscope and the polarization parameters can facilitate the quantitative detection of breast ductal carcinoma tissues at different stages.

The cutaneous microcirculation represents an index of the health status of the cardiovascular system. Conventional methods to evaluate skin microvascular function are based on measuring blood flow by laser Doppler in combination with reactive tests such as post-occlusive reactive hyperaemia (PORH). Moreover, the spectral analysis of blood flow signals by continuous wavelet transform (CWT) reveals nonlinear oscillations reflecting the functionality of microvascular biological factors, e.g. endothelial cells (ECs). Correlation mapping optical coherence tomography (cmOCT) has been previously described as an efficient methodology for the morphological visualisation of cutaneous micro-vessels. Here, we show that cmOCT flow maps can also provide information on the functional components of the microcirculation. A spectral domain optical coherence tomography (SD-OCT) imaging system was used to acquire 90 sequential 3D OCT volumes from the forearm of a volunteer, while challenging the micro-vessels with a PORH test. The volumes were sampled in a temporal window of 25 minutes, and were processed by cmOCT to obtain flow maps at different tissue depths. The images clearly show changes of flow in response to the applied stimulus. Furthermore, a blood flow signal was reconstructed from cmOCT maps intensities to investigate the microvascular nonlinear dynamics by CWT. The analysis revealed oscillations changing in response to PORH, associated with the activity of ECs and the sympathetic innervation. The results demonstrate that cmOCT may be potentially used as diagnostic tool for the assessment of microvascular function, with the advantage of also providing spatial resolution and structural information compared to the traditional laser Doppler techniques.

We present the development of an endoscopic ultrafast laser scalpel with improved miniaturized optics as a follow-up to our previous studies. We previously determined that the nonlinear susceptibility of ZnS crystal lenses at high pulse energies can limit the maximum energy reaching the tissue surface. Here, we improve the nonlinear properties of miniaturized optics using CaF2 in lieu of ZnS as a lens material to mitigate the problem of three-photon absorption at the high energies needed for rapid tissue ablation. We built and tested a miniaturized objective consisting of a CaF2 crystal lens pair to focus ultrashort laser pulses delivered through a large air core Kagome fiber to a 3.4 μm diffraction limited spot and scan the beam with a piezo-tube across a 100×100 μm^2 field of view (FOV). Negligible three-photon absorption and high transmission through the probe (>50%) allows for delivery of fluences >8.0 J/cm^2. The entire opto-mechanical system, enclosed within a 5-mm hypodermic tubing, can remove tissue at material removal rates (MRR) >0.5 mm^3/min in excised soft (porcine vocal folds) and hard (bovine rib bone) tissue samples. We found that MRRs could be increased by the optimized combination of piezo- and translation scanning parameters, providing clinically relevant tissue removal speeds. Further, we present the first ultrafast tissue ablation experiments in a live animal model (hamster cheek pouch) using a handheld surgical probe. Towards clinical acceptance, we designed an injection-molded enclosure to seal the 5-mm diameter opto-mechanical assembly and constituent wires into a sterilization-ready ergonomic handheld stylus tool.

Aperture synthesis techniques such as Interferometric Synthetic Aperture Microscopy (ISAM) and digital refocusing allow to restore Optical Coherence Tomography (OCT) images in out-of-focus regions and obtain increased spatial resolution. Since these techniques are phase sensitive, they require object stability during OCT data acquisition. Since these techniques are manipulate volumetric data as well, application of the techniques to in vivo measurements require some motion correction procedure. In this work we will show that some of these correction procedures can obliterate not only local phase variations caused by the objects motion during the acquisition but local phase variations caused by defocusing, thus erasing information necessary for OCT image restoration. Ways of overcoming the problem are presented and discussed.

Low-cost and high-performance 3-D light-sheet fluorescence imaging on pre-owned conventional microscopes Tingting Zhu, Xinlin Xie, Yao Yao, Dan Zhu2,* and Peng Fei1, 2, * 1. School of Optics and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074, China 2. Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China Abstract: light-sheet fluorescence microscopy (LSFM) is a promising imaging technique that enable imaging of samples in three dimensions at high speed and low phototoxicity1.LSFM is fundamentally characterized by its separate laser-sheet illumination path that provides optical sectioning of the samples and otherwise shows the same wide-field fluorescence detection with the conventional microscopes. Most of LSFM modalities are currently independent from either conventional microscopes, containing highly complicated setting with large form factors and high maintenance. Given the number of epifluorescence microscopes in service, a much easier way to access advanced LSFM imaging can be realized by creating a laser-sheet illumination and make use of existing conventional microscope for fluorescence readout. Here we present light-sheet imaging plugin (LIP) method that readily enables multi-dimensional, high spatiotemporal resolution imaging from single cells to whole organisms on an inverted microscope2.Furthermore, LIP can be also combined with microfluidics techniques to achieve high-throughput, high-resolution 3-D imaging/screening at a speeds up to 30 samples per seconds3.The ability to achieve high-speed, multi-dimensional imaging on a conventional microscope renders LIP device a valuable tool for many biomedical applications such as embryo development, tissue pathology and neuroscience. Furthermore, its compact add-on format allows the full use of pre-owned equipment and renovation at an affordable expense, which could substantially benefit smaller and less-funded departments / laboratories for not limiting the access to greater biological research in the absence of commercial light-sheet systems. Keywords: Light-sheet microscopy, light-sheet imaging plugin, microfluidics, 3-D biomedical imaging Reference: 1. Power R M, Huisken J. A guide to light-sheet fluorescence microscopy for multiscale imaging.[J]. Nature Methods, 2017, 14(4):360. 2. Guan Z, Lee J, Jiang H, et al. Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope[J]. Biomedical Optics Express, 2015, 7(1):194. 3. Jiang H, Zhu T, Zhang H, et al. Droplet-based light-sheet fluorescence microscopy for high-throughput sample preparation, 3-D imaging and quantitative analysis on a chip[J]. Lab on A Chip, 2017.

Two-photon excited fluorescence (TPEF) microscopy provides spectacular insights into visualization of cellular events within intravital tissue due to advantages of inherent sectioning ability, relatively deep optical penetration, and low optical damage. However, because the excitation spectrum of multiple fluorophores (FPs) are often separated, the spectral band of standard 100-fs Ti: Sapphire oscillators is not sufficiently board to efficiently excite multiple FPs. By pumping pulses from the 100-fs Ti: Sapphire oscillator through the highly nonlinear photonic crystal fibers (HNPCF), continuum pulses with 700-900 nm spectra covering common FPs can be generated due to enhanced self-phase modulation. We demonstrated that broadband fiber continuum combined with phase shaping technology can be ideal for multicolor TPEF microscopy. By phase shaping the continuum pulses, rapid selective excitation of specified FPs can be achieved to decrease the spectral crosstalk of multiple FPs in multicolor TPEF microscopy. We achieved selective excitation which allows in vivo imaging of four FPs in skin fold window chamber of mouse. The result shows that the selective excitation efficiency and contrast ratios are both high. Phase shaping selective excitation using fiber continuum is expected to optimally excite and separate multiple FPs when synchronously monitoring the dynamic characteristics of a variety of cells in tumor microenvironment. This work shows potential to promote multicolor two-photon fluorescence microscopy in the application for in vivo imaging.

Functional near-infrared spectroscopy (fNIRS) is a powerful clinical tool for monitoring hemoglobin concentration in brain tissues by analyzing absorption of scattered light. Since human brain is composed of multilayers including scalp, skull, and cerebral cortex, fNIRS signals need be analyzed with a multilayer tissue model. However, retrieving the optical properties of a multilayer tissue is often difficult because nonlinear fitting of absorption parameters from a scattered light signal by a tissue is ill-posed especially when the signal level is low. In this paper we introduce the cost function based masking technique for effective error minimization in the nonlinear fitting of fNIRS signals. We have shown that this method effectively reduces the influences of measurement errors with a newly defined cost function. Numerically simulated fNIRS data were generated for a two-layered tissue model and are used to extract the optical parameters of the two-layered tissue model. Accuracies of extracted parameters were compared with and without our proposed cost function.

Nanoparticle contrast agents for targeted imaging have widespread diagnostic applications with cellular resolution, specificity and selectivity for visualization and assessment of various disease processes. Of particular interest are gold nanoparticles owing to the tunability of the localised surface plasmon resonance (LSPR) and its relative inertness. Synthesizing gold nanoprobes in the near infrared (NIR) region is of particular interest in developing nanosensors due to the minimal light attenuation from biomolecules. The ability of plasmonic gold nanostars (GNS), with novel shape-dependent dual LSPR, to elicit signal contrast at NIR wavelengths is described here for multiple biomedical modalities. First, the surface enhanced Raman scattering (SERS) capability of these dual plasmonic GNS has been demonstrated to elicit high SERS enhancement factor (EF) of 2 x 10e7 with 785 nm excitation and the potential to elicit the highest SERS EF ever reported for gold nanoparticles, with further longer wavelength excitations at and beyond 1064 nm. We have also demonstrated the longer wavelength contrast imaging capability of GNS with photoacoustic imaging (PAI) and for photothermal therapy (PTT). GNS possess unique structural characteristics that impart superior optical properties resulting in higher photothermal efficiency. The photothermal capability of GNS was demonstrated in vivo with localized temperature rise of 9℃ in tumors when irradiated with a 1064 nm CW laser that resulted in significant tumor cell death. Since photothermal conversion is the optical process responsible for eliciting PA contrast and for PTT, this development represents a novel theranostic substrate to be used at 1064 nm excitation, a longer wavelength than the conventional clinical range. The ability of GNS to elicit signal contrast at NIR wavelengths has also been demonstrated for photothermal optical coherence tomography (PT-OCT). When irradiated with a 1064 nm continuous wave laser, GNS elicited photothermal contrast well beyond 2 mm, displaying great potential for deep tissue imaging. We have also recently obtained a European Commission grant worth €5.98M on developing, demonstrating and validating a novel GNS enhanced photoacoustic imaging platform which will be capable of tracking mesenchymal stem cells (MSC) and MSC-derived exosomes, at unprecedented depth and sensitivity.

It has been demonstrated that Mueller matrix imaging is capable of extracting the microstructures and optical properties of the tissue. We try to explain how different clearing agent affects the microstructure of the tissues during the optical clearing process using Mueller matrix analysis. We employ two type agents and record how the Mueller matrix elements vary with the immersion time, and then we compare the dynamic polarization information with the Monte Carlo simulations focusing on different possible clearing mechanisms. By analyzing the connection between Mueller matrix elements and the microstructural variation and optical features of tissues, we try to explain the microscopic phenomena induced by different agents. Then we simulated several possible cases of microstructural changes caused by tissue lesions, and then observe the influence of different agent on polarization imaging contrast by the simulation process of the possible clearing mechanisms. These research works not only confirm that Mueller matrix imaging combined with Monte Carlo simulation is potentially a powerful tool for monitoring and understanding tissue optical clearing, but also show how tissue clearing agents can improve the polarization identification and what type tissue can be improved clearer.

There is increasing interest in the three-dimensional visualization and quantification of cellular circuits in the brain and therefore optical clearing methods are highly in demand for brain imaging. In particular, clarification without membrane damage is required to image lipophilic tracer-labeled neural tracts. However, previously reported DiI-compatible optical clearing methods are relatively slow and can hinder transparency for imaging. Here, we present DAS, a new, convenient, inexpensive and reproducible aqueous clearing reagent that can efficiently clarify tissues with minimal volume enlargement and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues.

The main issue of epileptology is the elimination of epileptic events. This can be achieved by a system that predicts the emergence of seizures in conjunction with a system that interferes with the process that leads to the onset of seizure. The prediction of seizures remains, for the present, unresolved in the absence epilepsy, due to the sudden onset of seizures. We developed an algorithm for predicting seizures in real time, evaluated it and implemented it into an online closed-loop brain stimulation system designed to prevent typical for the absence of epilepsy of spike waves (SWD) in the genetic rat model. The algorithm correctly predicts more than 85% of the seizures and the rest were successfully detected. Unlike the old beliefs that SWDs are unpredictable, current results show that they can be predicted and that the development of systems for predicting and preventing closed-loop capture is a feasible step on the way to intervention to achieve control and freedom from epileptic seizures.

The presence of artifacts complicates analysis of physiological systems based on experimental time series. Aiming to increase the signal-to-noise ratio and to improve characterization of the system’s state, bad segments are simply removed from the experimental recording, and the latter may change correlation and other properties of the resulting dataset. Here we illustrate that in the case of positively correlated processes being typical for vascular dynamics, the authentic characterization of the system’s dynamics can be provided even under the condition of extreme data loss. Based on the cerebral blood flow (CBF) dynamics acquired with the laser speckle contrast imaging (LSCI) and the multiresolution analysis, we show insensitive changes of measures quantifying the system’s state with the amount of missed data for both, macro- and microcerebral circulation. We also demonstrate that these results do not significantly depend on the selected basic wavelet and the resolution level.

Authentic recognition of specific patterns of electroencephalograms (EEGs) associated with real and imagi- nary movements is an important stage for the development of brain-computer interfaces. In experiments with untrained participants, the ability to detect the motor-related brain activity based on the multichannel EEG processing is demonstrated. Using the detrended fluctuation analysis, changes in the EEG patterns during the imagination of hand movements are reported. It is discussed how the ability to recognize brain activity related to motor executions depends on the electrode position.

In this paper we study the spiking behaviour of a neuronal network consisting of 100 Rulkov elements coupled to each other with randomly chosen coupling strength. We find periodical grouping forming in the signal from all neurons in the network. We discovered the phenomenon of coherent resonance when signal-to-noise ration takes the maximum value at certain values of such parameters as number of neurons in the system, number of stimulated neurons, amplitude of external stimulus and amplitude of internal noise.

In this paper we study the correlation between the neurophysiological processes and personal characteristics arising in the processes of human higher mental functions. We find that the activity of the brain correlates with the results of psychological tests (according to the Cattell test). Experimental studies and math processing are described for operation design with the registration of human multi-channel EEG data in two phases (the processes of passive wakefulness (background) and special psychological testing (active phase)).

Using wavelet analysis of the signals of electrical brain activity (EEG), we study the processes of neural activity, associated with perception of visual stimuli. We demonstrate that the brain can process visual stimuli in two scenarios: (i) perception is characterized by destruction of the alpha-waves and increase in the high-frequency (beta) activity, (ii) the beta-rhythm is not well pronounced, while the alpha-wave energy remains unchanged. The special experiments show that the motivation factor initiates the first scenario, explained by the increasing alertness. Based on the obtained results we build the brain-computer interface and demonstrate how the degree of the alertness can be estimated and controlled in real experiment.

In the present paper the nonlinear association analysis of the EEG brain data in the process of bistable image perception are realized. Brain functional connectivity can be characterized by the temporal evolution of correlation between signals recorded from spatially-distributed regions. Numerous techniques were introduced for assessing this connectivity. Among nonlinear regression analysis methods, we chose a method introduced in the field of EEG analysis by Pijn, Lopes da Silva and colleagues, based on the fitting of a nonlinear curve by piecewise linear approximation, and more recently evaluated in a model of coupled neuronal populations. This method has some major advantages over other signal analysis methods such as coherence and cross-correlation functions because it can be applied independently of whether the type of relationship between the two signals is linear or nonlinear. In the capacity of bistable image we used a set of images based on a well-known bistable object, the Necker cube, as a visual stimulus. This is a cube with transparent faces and visible ribs. Bistability in perception consists in the interpretation of this 3D-object as to be oriented in two different ways, in particular, if the different ribs of the Necker cube are drawn with different intensity. It was shown that the structure of connections in the brain is different for cases without visual stimulation and with stimulation with the help of the Necker cube.

In present work we studied features of the human brain states classification, corresponding to the real movements of hands and legs. For this purpose we used supervised learning algorithm based on feed-forward artificial neural networks (ANNs) with error back-propagation along with the support vector machine (SVM) method. We compared the quality of operator movements classification by means of EEG signals obtained experimentally in the absence of preliminary processing and after filtration in different ranges up to 25 Hz. It was shown that low-frequency filtering of multichannel EEG data significantly improved accuracy of operator movements classification.

It has been demonstrated in many biomedical applications that polarization imaging is capable of probing the characteristic microstructural features of complex biological specimens quantitatively and non-invasively. In a recent study, we carried on backscattering Muller matrix imaging on living nude mice skin using oblique illumination by a 633nm LED light source. We quantitatively measured how the anisotropy properties of the living mice skin changes as functions of the UV exposure time. The time course features provide vital clue for the mechanism of UV damage and the effectiveness of sunscreen for reducing such damage. In this work, we report an upgraded system with LED light sources of five different colors ranging from blue to red. The system is calibrated by taking multi-color Mueller matrix images using a single set of rotating achromatic quarter-wave plates. In both in situ applications on living nude mice skin and ex vivo imaging of thick fresh tissue samples, we demonstrated that the multi-color polarized light backscattering measurements are able to reveal more details on the microstructure of the sample, particularly helpful in separating different effects due to photon scattering and propagation.

In the present research we studied the cognitive processes, associated with the perception of ambiguous images using the multichannel MEG recordings. Using the wavelet transformation, we considered the dynamics of the neural network of brain in different frequency bands, including high (up to 100 Hz) frequency gamma-waves. Along with the time-frequency analysis of single MEG traces, the interactions between remote brain regions, associated with the perception, were also taken into consideration. As the result, the new features of bistable visual perception were observed and the effect of image ambiguity was analyzed.

Problem of interaction between human and machine systems through the neuro-interfaces (or brain-computer interfaces) is an urgent task which requires analysis of large amount of neurophysiological EEG data. In present paper we consider the methods of parallel computing as one of the most powerful tools for processing experimental data in real-time with respect to multichannel structure of EEG. In this context we demonstrate the application of parallel computing for the estimation of the spectral properties of multichannel EEG signals, associated with the visual perception. Using CUDA C library we run wavelet-based algorithm on GPUs and show possibility for detection of specific patterns in multichannel set of EEG data in real-time.

The focal riddle for physicists and neuroscientists consists in disclosing the way microscopic scale neural interactions pilot the formation of the different activities revealed (at a macroscopic scale) by EEG and MEG equipments. In the current paper we estimate the degree of the interactions between the remote regions of the brain, based on the wavelet analysis of EEG signals, recorded from these brain areas. With the help of the proposed approach we analyze the neural interactions, associated with cognitive processes, taken place in human’s brain during the perception of visual stimuli. We show that neurons in the remote regions of brain interact with the different degree of intensity in the generation of different rhythms. In particular during the perception of visual stimuli strong interaction has been observed in β - frequency band while strong interaction in α - frequency band has been observed in resting state.

The spectra of luminescence of ZnCdS nanoparticles (ZnCdS NPs) were measured and analyzed in a wide temperature range: from room to human body and further to a hyperthermic temperature resulting in tissue morphology change. The results show that the signal of luminescence of ZnCdS NPs placed within the tissue is reasonably good sensitive to temperature change and accompanied by phase transitions of lipid structures of adipose tissue. It is shown that the presence of a phase transition in adipose tissue upon its heating (polymorphic transformations of lipids) leads to a nonmonotonic temperature dependence of the intensity of luminescence for the nanoparticles introduced into adipose tissue. This is due to a change in the light scattering by the tissue. The light scattering of adipose tissue greatly distorts the results of temperature measurements. The application of these nanoparticles is possible for temperature measurements in very thin or weakly scattering samples.

To minimise the influence of blood content on the fluorescence measurements in vivo, a fibre optical probe combining fluorescence and diffuse reflectance measurements was developed. For the inverse solution of the blood content recovery, a set of neural networks trained by the Monte Carlo generated learning set was used. An approach of fluorescence measurements triggered by simultaneous real-time measurements of blood content in living tissue during moderate changes in contact pressure of the optic probe is proposed. The method allows one to decrease the necessary pressure on the probe as well as increase the repeatability of the measurements. The developed approach was verified in a series of experiments on volunteers with fluorescence excitation at 365 nm and 450 nm. The proposed technology is of particular interest in the development of new fluorescence-based optical biopsy systems.

To monitor skin microvascular dysfunction of alloxan-induced type 1 diabetic mice model. In this work, we used laser speckle contrast imaging and hyperspectral imaging through in vivo skin optical clearing method to simultaneously monitor the noradrenaline-induced response of microvascular blood flow and blood oxygen with the development of diabetes. The main results showed that venous and arterious blood flow steadily decreased without recovery after injecting noradrenaline (NE), furthermore the influence of NE-induced arterious blood oxygen response greatly decreased, especially for 2-weeks and 4-weeks diabetic mice. This study demonstrated that skin microvascular function was a potential research biomarker for early warning in the occurrence and development of diabetes. And it provides a feasible solution to realize visualization of cutaneous microvessels for monitoring microvascular reactivity.

In this paper, we investigate the problem of identification of patterns on magnetoencephalography signals of a brain associated with human movements. The design of registration of experimental data during magnetoencephalography (MEG) is developed and described. Consecutive imaginary movements of the hands and legs of the person are chosen as the basic movements. We solve the problem of recognition and classification of patterns using artificial neural networks. For a multilayer perceptron, good results of recognition of patterns of brain activity associated with different types of motion have been obtained.

In-vivo optical imaging method provides information about the anatomical structures and function of tissues ranging from single cell to entire organisms. Dynamic Fluorescent Imaging (DFI) is used to examine dynamic events related to normal physiology or disease progression in real time. In this work we improve this method by using factor analysis (FA) to automatically separate overlying structures.The proposed method is based on a previously introduced Transcranial Optical Vascular Imaging (TOVI), which employs natural and sufficient transparency through the intact cranial bones of a mouse. Fluorescent image acquisition is performed after intravenous fluorescent tracer administration. Afterwards FA is used to extract structures with different temporal characteristics from dynamic contrast enhanced studies without making any a priori assumptions about physiology. The method was validated by a dynamic light phantom based on the Arduino hardware platform and dynamic fluorescent cerebral hemodynamics data sets. Using the phantom data FA can separate various light channels without user intervention. FA applied on an image sequence obtained after fluorescent tracer administration is allowing extracting valuable information about cerebral blood vessels anatomy and functionality without a-priory assumptions of their anatomy or physiology while keeping the mouse cranium intact. Unsupervised color-coding based on FA enhances visibility and distinguishing of blood vessels belonging to different compartments. DFI based on FA especially in case of transcranial imaging can be used to separate dynamic structures.

Needle-free jet injection is a transdermal drug delivery technique wherein a liquid drug is pressurized, and ejected through a ~200 μm orifice at high speed (~200 m/s). The resulting fluid jet can rapidly penetrate through the skin, and disperse in the underlying tissue at a speed-related depth. Our electronically controllable injection systems uniquely offer the possibility of depth-control during injection. To this end, we have developed a spatially-resolved diffuse imaging technique to provide an estimate of the injection depth. An injection system was constructed to couple a collimated laser beam into the fluid jet as it was ejected through the orifice. During an injection, the penetration of the jet into a tissue-mimicking phantom eroded an unobstructed optical path for the laser beam before it impinged on the scattering medium at the bottom of the hole. This resulted in a pattern of backscattered light around the injection site that varied as a function of injection depth. We performed laser-coupled injections into a light-scattering polyacrylamide gel, while recording high-speed videos of the diffuse light exiting from the side and surface of the phantom. The centroid of the light distribution exiting from the side of the phantom was used as the estimate for the injection depth. A strong correlation was found between the depth of the centroid and the surface light profile, showing that it is possible to infer the injection depth from the spatial distribution of light around the injection site alone.

The conditions of selective laser melting (SLM) of tissue engineering scaffolds affect cell response and must be engineered to support cell adhesion, proliferation, and differentiation. In the present study, the influence of additional heating during SLM process on stem cell viability near biopolymer matrix reinforced by nanoceramics additives was carried out. We used the biocompatible and bioresorbable polymers (polyetheretherketone /PEEK/ and polycaprolactone /PCL/) as a matrix and nano-oxide ceramics - TiO₂, A_l2O₃, ZrO₂, FexOy and/or hydroxyapatite as a basis of the additives. The rate of pure PEEK and PCL bio-resorption and in mixtures with nano oxides on the matrix was studied by the method of mass loss on bacteria of hydroxylase and enzyme complex. The stem cellular morphology, proliferative MMSC activity, and adhesion of the 2D and 3D nanocomposite matrices were the subjects of comparison. Medical potential of the SLS/M-fabricated nano-oxide ceramics after additional heating as the basis for tissue engineering scaffolds and cell targeting systems were discussed.