We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65  W/cm ² at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.

Focusing light inside highly scattering media is a challenging task in biomedical optical imaging, manipulation, and therapy. A recent invention has overcome this challenge by time reversing ultrasonically encoded diffuse light to an ultrasound-modulated volume inside a turbid medium. In this technique, a photorefractive (PR) crystal or polymer can be used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the PR response time of the PR material, is usually used to encode the diffuse light. This long burst results in poor focusing resolution along the acoustic axis. In this work, we propose to use two intersecting ultrasound beams, emitted from two ultrasonic transducers at different frequencies, to modulate the diffuse light at the beat frequency within the intersection volume. We show that the time reversal of the light encoded at the beat frequency can converge back to the intersection volume. Experimentally, an acoustic axial resolution of ∼1.1  mm was demonstrated inside turbid media, agreeing with theoretical estimation.

Depth-sensitive fluorescence spectroscopy in a two-layered epithelial tissue phantom was demonstrated using a cone shell configuration implemented by an axicon lens and a fiber assembly including five rings of collection fibers. Each collection fiber ring was located at a different radial distance away from the center, for which fluorescence measurements from all rings showed a larger range of sensitivities to the top and bottom layers compared to the previously reported cone shell configuration. Furthermore, multiple fluorescence spectra corresponding to a range of successive targeted depths can be obtained simultaneously, which shortened data acquisition dramatically and eliminated the mechanical moving components required for depth-sensitive optical measurements in the previous setup. This new setup therefore would be preferred in a clinical setting.

Cherenkov radiation is induced when charged particles travel through dielectric media (such as biological tissue) faster than the speed of light through that medium. Detection of this radiation or excited luminescence during megavoltage external beam radiotherapy (EBRT) can allow emergence of a new approach to superficial dose estimation, functional imaging, and quality assurance for radiation therapy dosimetry. In this letter, the first in vivo Cherenkov images of a real-time Cherenkoscopy during EBRT are presented. The imaging system consisted of a time-gated intensified charge coupled device (ICCD) coupled with a commercial lens. The ICCD was synchronized to the linear accelerator to detect Cherenkov photons only during the 3.25-μs radiation bursts. Images of a tissue phantom under irradiation show that the intensity of Cherenkov emission is directly proportional to radiation dose, and images can be acquired at 4.7  frames/s with SNR<30 . Cherenkoscopy was obtained from the superficial regions of a canine oral tumor during planned, Institutional Animal Care and Use Committee approved, conventional (therapeutically appropriate) EBRT irradiation. Coregistration between photography and Cherenkoscopy validated that Cherenkov photons were detected from the planned treatment region. Real-time images correctly monitored the beam field changes corresponding to the planned dynamic wedge movement, with accurate extent of overall beam field, and expected cold and hot regions.

The versatility and real-time imaging capability of commercial linear array transducers make them widely used in clinical ultrasound and photoacoustic imaging. However, they often suffer from limited detection view. For instance, acoustic waves traveling at a grazing angle to the transducer surface are difficult to detect. In this letter, we propose a simple and easy approach to ameliorate this problem by using a 45-deg acoustic reflector. The reflector forms a virtual array that is perpendicular to the physical array, thereby doubling the detection coverage. The improvement in image quality in photoacoustic tomography was demonstrated through a hair phantom, a leaf skeleton phantom, and an ex vivo mouse ear experiment.

Biophotonics is now a major area of activity and growth in Israeli academia, hospitals, and industry. Leading biophotonics companies have been established and operate in Israel. The entrepreneurial nature of the country stimulates translational research in this field, and a large number of Israeli start-up companies are developing cutting-edge biophotonics technologies.

We present an optoelectrical device capable of in vitro optical stimulation and electrophysiological recording. The device consists of an array of micropixellated InGaN light-emitting diodes coupled to a custom-made ultrathin planar microelectrode array. Cells can be cultured directly on the chip for short- and long-term electrophysiological experiments. To show the functionality of the device, we transfected a cardiomyocyte-like cell line (HL-1) with a light-sensitive protein channelrhodopsin. We monitored action potentials of individual, spontaneously beating, HL-1 cells growing on the chip by extracellular electrical recordings. On-chip optical stimulation was demonstrated by triggering network activity in a confluent HL-1 cell culture and visualized by calcium imaging. We see the potential of our system for electrophysiological experiments with optogenetically modified cells. Optical stimulation can be performed directly on the chip without additional optical components or external light sources.

A new skin-imaging method, which was developed recently, gives an informative image data about skin moles by collecting scattered polarized light reflected from their tissue area at different wavelengths and polarizations. This method is called as optical rotational spectropolarimetric imaging (ORSI), which scans the polarization states by continuously rotating a linearly polarized light incident on the lesion and collecting the reflected sequence of images. A novel method to distinguish cancerous from benign moles by analyzing the images obtained using this imaging system is proposed. The proposed method performs an automatic examination of the polarized images according to characteristics such as their cross-image local contrasts, large-scale homogeneity, border disorder, and asymmetry. The pilot study was conducted with 10 subjects, in which two ORSI image sequences at two different wavelengths were taken for each subject. Results show good separation between cancerous and benign moles and between benign moles with high dysplasia.

Vascular and microvascular anastomoses are critical components of reconstructive microsurgery, vascular surgery, and transplant surgery. Intraoperative surgical guidance using a surgical imaging modality that provides an in-depth view and three-dimensional (3-D) imaging can potentially improve outcome following both conventional and innovative anastomosis techniques. Objective postoperative imaging of the anastomosed vessel can potentially improve the salvage rate when combined with other clinical assessment tools, such as capillary refill, temperature, blanching, and skin turgor. Compared to other contemporary postoperative monitoring modalities—computed tomography angiograms, magnetic resonance (MR) angiograms, and ultrasound Doppler—optical coherence tomography (OCT) is a noninvasive high-resolution (micron-level), high-speed, 3-D imaging modality that has been adopted widely in biomedical and clinical applications. For the first time, to the best of our knowledge, the feasibility of real-time 3-D phase-resolved Doppler OCT (PRDOCT) as an assisted intra- and postoperative imaging modality for microvascular anastomosis of rodent femoral vessels is demonstrated, which will provide new insights and a potential breakthrough to microvascular and supermicrovascular surgery.

We show that phase-conjugation by three-wave mixing allows turbidity suppression through biological tissues with thicknesses up to 5 mm, at a near-infrared wavelength included in the therapeutic window. Because of the ultrafast character of the imaging process, a motion of the tissue, which mimics in vivo imaging, can be exploited to significantly improve the signal-to-noise ratio and the resolution of the restored images.

Fractional laser ablation is one of the relatively safe and minimally invasive methods used to administer micro- and nanoparticles into the skin at sufficiently large depth. In this article, we present the results of delivery of TiO ₂ nanoparticles and Al ₂ O ₃ microparticles into skin. Fractional laser microablation of skin was provided by a system based on a pulsed Er:YAG laser with the following parameters: the wavelength 2940 nm, the pulse energy 3.0 J, and the pulse duration 20 ms. Ex vivo and in vivo human skin was used in the study. The suspensions of titanium dioxide and alumina powder in polyethylene glycol with particle size of about 100 nm and 27 μm, respectively, were used. In the ex vivo experiments, reflectance spectra of skin samples with administered particles were measured and histological sections of the samples were made. In the in vivo experiment, reflectance spectroscopy, optical coherence tomography, and clinical photography were used to monitor the skin status during one month after suspension administering. It is shown that particles can be delivered into dermis up to the depth 230 μm and distributed uniformly in the tissue. Spectral measurements confirm that the particles stay in the dermis longer than 1 month.

Morphological changes of the adipose tissue at phototreatment are studied in vitro using optical coherence tomography. The 200 to 600 μm fat tissue slices are used in the experiments. The observed change in the tissue structure was associated with fat cell lipolysis and destruction caused by the photodynamic effect. It is found that overall heating of a sample from room to physiological temperature leads to deeper and faster morphology tissue changes if other processing conditions are kept constant. These data support the hypothesis that photodynamic/photothermal treatment induces fat cell lipolysis during some period after treatment.

Most methods for measuring light–tissue interactions focus on the volume reflectance while very few measure the transmission. We investigate both diffusion reflection and diffuse transmission at all exit angles to receive the full scattering profile. We also investigate the influence of blood vessel diameter on the scattering profile of a circular tissue. The photon propagation path at a wavelength of 850 nm is calculated from the absorption and scattering constants via Monte Carlo simulation. Several simulations are performed where a different vessel diameter and location were chosen but the blood volume was kept constant. The fraction of photons exiting the tissue at several central angles is presented for each vessel diameter. The main result is that there is a central angle that below which the photon transmission decreased for lower vessel diameters while above this angle the opposite occurred. We find this central angle to be 135 deg for a two-dimensional 10-mm diameter circular tissue cross-section containing blood vessels. These findings can be useful for monitoring blood perfusion and oxygen delivery in the ear lobe and pinched tissues.

The disease- and drug-related bone disorders are rapidly increasing in the population. It is previously reported that anti-epileptic drugs (AEDs) may cause osteopenia, osteoporosis, and fractures in epilepsy patients. However, it cannot be determined whether the bone disorders in epileptic patients are due to AED therapy and/or to epilepsy and epileptic seizures. There is no study in the literature which investigates the sole effects of epilepsy and epileptic seizures on bone tissues. The current study provides the first report on determination of the possible effects of epilepsy and epileptic seizures on long bone tissues. Wistar Albino Glaxo/Rijswijk rats, which are accepted as genetic rat models for human absence epilepsy, were compared with the healthy Wistar rats to get information about the sole effects of epilepsy and epileptic seizures on bones. Cortical regions of tibia and femur bones were studied by Fourier transform infrared microspectroscopy (FTIRM). According to FTIRM parameters, variation on bone mineral and matrix composition, including decreased mineral content, decreased collagen cross-links, increased carbonate substitution, and larger crystals in epileptic group compared to the healthy one, show severe effects of epilepsy and seizures on bone tissues for the first time.

Treating cancer is one of the major challenges of modern medicine. Since mice models are an important tool in cancer treatment research, it is required to assess murine tumor development. Existing methods for investigating tumor development are either high cost and limited by their availability or suffer from low accuracy and reproducibility. In order to overcome these drawbacks, thermography may be used. DA3 breast cancer carcinoma tumors in 12  Balb/c mice were thermally imaged and monitored for a period of several weeks. Eight mice were treated with diffusing alpha emitters radiation therapy (DaRT) wires, while four were treated with inert wires. For large tumors, the area was estimated by analyzing thermal images and was found to be in correlation with manual caliper measurements. In addition, the correlation between tumor area and relative temperatures was calculated and compared to previous works. Temperature differences were larger for tumors treated with DaRT wires than tumors with inert wires. These correlations can be used to assist in tumor size estimation and reveal information regarding its metabolic state. Overall, thermography was shown to be a promising tool for assessing tumor development with the additional advantages of being nonradiative and potentially providing indication of intratumoral biological processes.

A simple optical tweezers design is proposed to manipulate particles in the axial direction and estimate particle position with nanometer sensitivity. Balb3T3 cell is probed using two different-sized particles, and the localized cell stiffness is evaluated using Hertz model. A series of experiments are performed to obtain the necessary parameters for the cell stiffness computation: particle displacement, trapping stiffness, force exertion, and cell deformation. The computed cell stiffness measurements are 17 and 40 Pa using 4 μm- and 2 μm-sized particles, respectively. Results suggest that the proposed optical tweezers scheme can measure the stiffness of a particular cell locale using Hertz model, offering insights about how cells respond to outside mechanical stimulus.

There is currently a need for a safe and effective way to detect and diagnose early stages of childhood caries. A multimodal optical clinical prototype for diagnosing caries demineralization in vivo has been developed. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and high-contrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. When a suspicious region of demineralization is located, the device also performs dual laser fluorescence spectroscopy using 405- and 532-nm laser excitation. An autofluorescence ratio of the two excitation lasers is computed and used to quantitatively diagnose enamel health. The device was tested on five patients in vivo as well as on 28 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images that highlighted the lesions identified by the clinicians. The autofluorescence spectroscopic ratios obtained from the extracted teeth successfully quantitatively discriminated between sound and demineralized enamel.

We applied coherent phase microscopy to develop a method of quantitative evaluation of functional state of eukaryotic cells using the coordinates of characteristic points (CP) in the functions of the phase volume W and area S . In a fragment of a single cell image (HCT116 human colon carcinoma cell line) with detectable nucleolus, the values of the phase thickness, area, and volume were calculated. These values dramatically changed within the initial minutes of cell exposure to the transcriptional inhibitor actinomycin D . The positions of CP in the graphs of S and W functions allowed for monitoring the time-dependent decrease of nucleolar contrast, a major optical hallmark of “nucleolar stress.” Given that the area and volume functions reflect optical heterogeneity of the cell and are independent of its optical model, these functions can be applicable as general mathematical tools for the analysis of cell morphology and physiology.

We present a method for adding molecular specificity to wide-field interferometric phase microscopy (IPM) by recording the phase signatures of gold nanoparticles (AuNPs) labeling targets of interest in biological cells. The AuNPs are excited by time-modulated light at a wavelength corresponding to their absorption spectral peak, evoking a photothermal (PT) effect due to their plasmonic resonance. This effect induces a local temperature rise, resulting in local refractive index and phase changes that can be detected optically. Using a wide-field interferometric phase microscope, we acquired an image sequence of the AuNP sample phase profile without requiring lateral scanning, and analyzed the time-dependent profile of the entire field of view using a Fourier analysis, creating a map of the locations of AuNPs in the sample. The system can image a wide-field PT phase signal from a cluster containing down to 16 isolated AuNPs. AuNPs are then conjugated to epidermal growth factor receptor (EGFR) antibodies and inserted to an EGFR-overexpressing cancer cell culture, which is imaged using IPM and verified by confocal microscopy. To the best of our knowledge, this is the first time wide-field interferometric PT imaging is performed at the subcellular level without the need for total internal reflection effects or scanning.

A long-term live-imaging workstation to follow the development of cultured neurons during the first few days in vitro (DIV) is developed. In order to monitor neuronal polarization and axonal growth by live imaging, we built a micro-incubator system that provides stable temperature, pH, and osmolarity in the culture dish under the microscope, while preserving environment sterility. We are able to image living neurons at 2 DIVs for 48 h with a temporal resolution of one frame for every 2 min. The main features of this system are its ability to adapt to every cell-culture support, to integrate in any optical microscope, because of the relatively small dimensions (9.5×6.5×2.5  cm ) and low weight of the system (<200  g ), and to monitor the physiological parameters in situ. Moreover, we developed an image-analysis algorithm to quantify the cell motility, in order to characterize its complex temporal-spatial pattern. The algorithm applies morphological image processing operations on the temporal variations occurring in the inspected region of interest. Here, it is used to automatically detect cellular motility in three distinct morphological regions of the neurons: around the soma, along the neurites, and in the growth cone.

Although there has been great interest in laser heating for bonding of surgical incisions in tissues, it has not gained wide acceptance by surgeons. We argue that the main obstacle has been the lack of temperature control, which may lead to a weak bonding. We previously developed a laser bonding system based on two infrared transmitting AgBrCl fibers, one for laser heating and one for temperature control. In view of the inherent limitations of such systems observed in many animal experiments, we developed an improved system based on a single infrared fiber. Besides the decreased dimensions, this system offers many advantages over the two-fiber system. It is less sensitive to accuracy of height and tilt of the fiber distal tip above the tissue, ensuring more accurate heating that can potentially lead to stronger bonding with minimal thermal damage. The system is successfully tested in the soldering of 15 corneal incisions, ex vivo. Histopathology shows little thermal damage and good wound apposition. The average burst pressure is 100±30  mm Hg . These findings indicate the usefulness of the system for ophthalmic surgery as well as other surgical procedures, including endoscopic and robotic surgery.

Damage of collagen fibers in tendons is often directly related to changes in a tendon’s mechanical properties. Direct quantitative elasticity measurement of tendons will provide important information in tendon dysfunction diagnosis and treatment assessment. A feasibility study of quantifying the mechanical properties of a degenerated tendon model by a nondestructive imaging modality, which combines optical coherence elastography and acoustic radiation force (ARF) method, is presented. The degenerated tendon model was produced by the partial degradation of chicken tendons through incubation with collagenase at different concentrations and incubation times. A 30-kHz longitudinal ultrasound transducer was used to provide an ARF signal, which was detected by an ultra-high sensitive phase sensitive optical coherence tomography (PhS-OCT) system. The experimental results demonstrate that the combination of ARF method and PhS-OCT can measure the elasticity of tendon quantitatively. The corresponding changes in tendon elasticity due to the application of collagenase have been revealed by this new imaging modality. This method can potentially be used in the assessment of tissue engineering products and in the diagnosis and treatment progression of tendon diseases.

The Cramer-Rao lower bound (CRLB) on localization precision of unbiased estimators is analyzed for stochastic optical localization nanoscopy that localizes emitters frame by frame independently. It is found that the CRLB is a function of the mean number of detected photons per emitter, signal to Poisson noise ratio, signal to Gaussian noise ratio, point spread function (PSF), pixel size, and relationship of emitter locations. With a slight and practical approximation, effect of Gaussian noise is equivalent to increasing the mean photon count of Poisson noise by a number equal to the variance of Gaussian noise. Numerical examples demonstrate that the CRLB of emitters located on a curve increase fast as the distance of adjacent emitters increases. The mean CRLB of randomly uniformly distributed emitters in both two-dimensional and three-dimensional imaging increases exponentially fast as the emitter density increases. The effects of PSF, standard deviation of PSF, mean number of detected photons per emitter, signal to noise ratio, axial thickness, and pixel size on the CRLB are also numerically investigated. The analytical and numerical results provide a guideline for the design of location estimators and a benchmark for the achievable localization precision of stochastic optical localization nanoscopy.

Blood flow velocity and red blood cell concentration are of vital importance in assessing tissue microcirculation. Laser speckle contrast analysis is being considered as a promising tool in the qualitative assessment of flow velocity as well as scatterer concentration in different body fluids, though the quantification part still remains challenging. The fractal-based spatial correlation analysis of speckle flow images along with the corresponding contrast analysis for the quantitative assessment of flow and scatterer concentration is investigated. In this study, phantom body fluid solution (intralipid 20%) of different concentrations is pumped at different flow rates through the designed flow channel using a syringe pump and the corresponding speckle images are acquired. The fractality of the acquired speckle images in response to the changes in concentration of the fluid as well as the variations in fluid flow is analyzed along with the corresponding contrast-based analysis. Following this qualitative analysis, an experimental model is attempted toward quantification of these parameters from a single acquired speckle image by considering the contrast and fractality changes together.

This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.

An on-chip spectral surface plasmon resonance (SPR) biosensor with three parallel channels has been built for biomolecule detections. It is capable of updating intensity baselines to suppress errors induced by light source instability. In order to minimize structure and reduce positional regression uncertainty, the scan operations are achieved by timely changing optical media instead of conventional mechanical scan. The detection results of human immunoglobulin G (hIgG) demonstrated that the baseline updating improves the limitation of detection of hIgG concentration from ∼86.1 to ∼17.4  ng/mL.

The backward second harmonic generation (SHG) in mouse tissues is studied with a confocal multiphoton microscopy system. The total backward collected SHG (B-SHG) consists of the backward generated SHG and the backward-scattered forward-generated SHG (BS-SHG), which can be modeled by a Gaussian and a uniform distribution, respectively, at the confocal pinhole plane. By varying the pinhole size with a series of collection fibers, the proportion of the BS-SHG to the B-SHG and the proportion of BS-SHG to the forward generated SHG can be obtained. The approach is first validated by Monte Carlo simulation. It is then applied to two types of mouse tissues: mouse tail tendon and Achilles tendon. It is found that the BS-SHG contributes less to the B-SHG for the tail tendon than Achilles tendon with thicknesses of ∼300  μm . With the thickness of the Achilles tendon tissue increased to 1000 μm but the focal plane kept at the same depth, as high as ∼10% of the total forward SHG is backscattered and collected. The results indicate that BS-SHG may not be the major source of B-SHG in the tail tendon, but it may be the major source in the Achilles tendon. These methods and results provide a noninvasive method and supporting information for investigating the generation mechanism of SHG and help with optimizing backward SHG microscopy and spectroscopy measurements.

Components present in the acellular fraction of blood influence the blood cell survival and function and the response to biotic and abiotic factors. Human plasma and sera have been used as therapeutic agents and are known to increase cell survival. White blood cells in normal blood are exposed to plasma components in vivo, but the effect of such plasma components in vitro on adherent peripheral blood mononuclear cells (PBMCs) that includes monocytes has not been fully investigated. We cultured human PBMCs with autologous plasma and observed structural variation due to plasma addition in PBMCs along with increased cell survival. Light microscopy of the cells showed increased granularity in plasma-treated cells. Fourier transform infrared (FTIR) spectroscopy was used to elucidate the possible mechanism by studying the changes in the biochemical composition of the cells that explained the observations. FTIR spectroscopy of plasma-treated cells show altered spectral pattern in the mid-IR region, indicating increased phospholipid levels. Heat-stable components in the plasma possibly increase the differentiation of PBMCs, as evident by increased phospholipid metabolism. The data suggest that plasma-stimulated membrane biogenesis may contribute to PBMC survival by inducing them to differentiate into antigen presenting cells (APCs) like macrophages and dendritic cells.

We have developed a reflection-based capability of the RetCam® platform, an FDA-cleared pediatric retinal-imaging instrument, for the purpose of measuring macular pigment levels as well as their spatial distributions in infants and children. Our modifications include narrow-band blue-wavelength excitation of the macular pigment absorption in combination with spectrally selective blue-wavelength readout of the reflection signals received by the instrument’s CCD detector array. Furthermore, an algorithm is developed that allows the computation of optical density maps for the macular pigment relative to peripheral retinal areas. This made it possible for the first time to directly measure macular pigment levels and their spatial features in the developing human retina. In contrast to adults, infants with measurable pigment levels had almost exclusively a narrow, circularly symmetric, pigment distribution. The described methodology holds promise for future investigations into the role of macular pigment in the developing human retina and the effect of dietary interventions in diseases resulting from a lack of normal carotenoid levels.

A new imaging technique which enables near real-time multispectral acquisition of the so-called degree of polarization (DOP) in polarimetry using incoherent white light is described. The experimental setup allows the interactive and dynamic acquisition of DOP for all the possible elliptic polarization states. In such a way, a complete chart of light-matter interaction can be obtained and besides many structure details can be enhanced. Thus, we present the calibration and first images acquired with this system. The particular characteristics of this setup allow it to be the perfect candidate for in vivo as well as ex vivo medical applications.

Transcranial direct current stimulation (tDCS) of the human sensorimotor cortex during physical rehabilitation induces plasticity in the injured brain that improves motor performance. Bi-hemispheric tDCS is a noninvasive technique that modulates cortical activation by delivering weak current through a pair of anodal–cathodal (excitation–suppression) electrodes, placed on the scalp and centered over the primary motor cortex of each hemisphere. To quantify tDCS-induced plasticity during motor performance, sensorimotor cortical activity was mapped during an event-related, wrist flexion task by functional near-infrared spectroscopy (fNIRS) before, during, and after applying both possible bi-hemispheric tDCS montages in eight healthy adults. Additionally, torque applied to a lever device during isometric wrist flexion and surface electromyography measurements of major muscle group activity in both arms were acquired concurrently with fNIRS. This multiparameter approach found that hemispheric suppression contralateral to wrist flexion changed resting-state connectivity from intra-hemispheric to inter-hemispheric and increased flexion speed (p<0.05 ). Conversely, exciting this hemisphere increased opposing muscle output resulting in a decrease in speed but an increase in accuracy (p<0.05 for both). The findings of this work suggest that tDCS with fNIRS and concurrent multimotor measurements can provide insights into how neuroplasticity changes muscle output, which could find future use in guiding motor rehabilitation.

A noninvasive, quick, reliable, and relatively cheap procedure for the diagnosis of onychomycosis is put forward. It is known that a nail may show an abnormal appearance, although only 50% of all the nails having such an appearance may owe it to the presence of onychomycosis; hence, adequate diagnosis of nail disease is needed for appropriate prescription of medication and treatment of the nail. In order to contribute to the process of improvement in the diagnosis, a procedure based on the analysis of medium-range infrared images is presented in which it is possible to observe energy changes mostly due to the changes in emissivity of the nail. As a nail is more affected by onychomycosis, such changes become more intense. Also, it was found that a nail without onychomycosis has a lower temperature than toe skin, but has a higher emission of energy. Fifty percent of the ailments that may a cause a fingernail or toenail to have an abnormal appearance are not considered in the present work.

A three-dimensional stereoscopic imaging modality (3D-SIM) based on a single optical channel and detector was developed to overcome some of the limitations of conventional 3D-SIM. It produces 3-D stereoscopic images by adjusting the angle of a transparent rotating deflector (TRD) to generate disparity between left and right images. The angular effect of the TRD was demonstrated to investigate the feasibility of the proposed method in 3-D stereoscopic image generation. Results indicate that image disparity increased as a function of the rotation angles of the TRD, while maintaining adequate 3-D perception. These results are expected to facilitate the practical use of a 3D-SIM in medicine.

Use of near-infrared (NIR) structured illumination technique has recently received great interest in biomedical research and clinical studies because of its ability to perform wide-field imaging and quantitatively map changes in tissue hemodynamic properties and morphological features in a noncontact and scan-free fashion. We report on the feasibility of using the same to quantitatively monitor and map changes in brain optical properties and physiological parameters pre- and post-closed head injury in a mouse model. Five anesthetized male mice underwent head injury by weight-drop model using a ∼50-g cylindrical metal object falling from a height of 90 cm onto the intact scalp. During experiments, NIR structured illumination was projected on the mouse head at two spatial frequencies and six different NIR wavelengths. A CCD camera positioned perpendicular to the head recorded the diffuse-reflected light. Computer analysis performed off-line on the captured data reveals spatiotemporal changes in the distribution of brain tissue absorption and reduced scattering coefficients. Using Beer’s law and Mie theory, hemodynamic (hemoglobin, oxygen saturation, and lipids) and morphological (scattering amplitude and power) changes up to 1-h post-trauma were observed in comparison with baseline measurements. Functional maps of different brain properties were also generated. Following injury, we found difference in both brain hemodynamic and morphologic properties with respect to baseline levels, where in some properties, this difference was considered statistically significant. Specifically, a t -test indicates a substantial decrease in oxyhemoglobin (HbO₂) concentration and tissue oxygen saturation (StO₂) post-injury (p < 0.01 and p < 0.001, respectively). Overall, our preliminary results demonstrate the potential application of NIR structured illumination technique to track and spatially map changes in intact mouse brain pathophysiological parameters following head injury.

Second-harmonic generation (SHG) microscopy has intrinsic contrast for imaging fibrillar collagen and has shown great promise for disease characterization and diagnostics. In addition to morphology, additional information is achievable as the initially emitted SHG radiation directionality is related to subresolution fibril size and distribution. We show that by two parameter fittings, both the emission pattern (F _SHG /B _SHG ) _creation and the reduced scattering coefficient μ′_s , can be obtained from the best fits between three-dimensional experimental data and Monte Carlo simulations. The improved simulation framework accounts for collection apertures for the detected forward and backward components. We apply the new simulation framework to mouse tail tendon for validation and show that the spectral slope of μ′_s obtained is similar to that from bulk optical measurements and that the (F _SHG /B _SHG ) _creation values are also similar to previous results. Additionally, we find that the SHG emission becomes increasingly forward directed at longer wavelengths, which is consistent with decreased dispersion in refractive index between the laser and SHG wavelengths. As both the spectral slope of μ ′ _s and (F _SHG /B _SHG ) _creation have been linked to the underlying tissue structure, simultaneously obtaining these parameters on a microscope platform from the same tissue provides a powerful method for tissue characterization.

Recently, we presented an add-on to a photoacoustic (PA) computed tomography imager that permits the simultaneous imaging of ultrasound (US) transmission parameters such as the speed of sound (SOS), without additional measurements or instruments. This method uses strong absorbers positioned outside the object in the path of light for producing laser-induced US to interrogate the object in a conventional PA imager. Here, we investigate the feasibility of using this approach, first with PA to pin-point the location of photothermal therapeutic agents and then with serial SOS tomograms to image and monitor the resulting local temperature changes when the agents are excited with continuous wave (CW) light. As the object we used an agar-based tissue-mimicking cylinder carrying beads embedded with different concentrations of gold nanospheres. PA and SOS tomograms were simultaneously acquired as the gold nanospheres were photothermally heated using a 532-nm CW laser. In a first approximation, using the relation between SOS of water and temperature, the SOS tomograms were converted into temperature maps. The experimental results were verified using simulations: Monte Carlo modeling of light propagation through a turbid medium and using the obtained absorbed energy densities in heat diffusion modeling for spatial temperature distribution.

We developed a three-beam Doppler optical coherence tomography (OCT) system that enables measurement of the velocity vector of moving particles in three-dimensions (3-D). The spatial orientation as well as the magnitude of motion can be determined without prior knowledge of the geometry of motion. The system combines three spectral-domain OCT interferometers whose sample beams are focused at the sample by a common focusing lens at three different angles. This provides three spatially independent velocity components simultaneously from which the velocity vector can be reconstructed. We demonstrate the system in a simple test object (rotating disc), a flow phantom, and for blood flow measurements in the retina of a healthy human subject. Measurements of blood flow at a venous bifurcation achieve a good agreement between in- and outflow and demonstrate the reliability of the method.

Raman spectroscopy can be used for analysis of objects by detecting the vibrational spectrum using label-free methods. This imaging method was applied to analysis of peripheral nerve regeneration by examining the sciatic nerve in vitro and in vivo. Raman spectra of intact nerve tissue had three particularly important peaks in the range 2800−3000  cm⁻¹ . Spectra of injured sciatic nerves showed significant changes in the ratio of these peaks. Analysis of cellular spectra suggested that the spectrum for sciatic nerve tissue reflects the axon and myelin components of this tissue. Immunohistochemical analysis showed that the number of axons and the myelinated area were reduced at 7 days after injury and then increased by 28 days. The relative change in the axon to myelin ratio showed a similar initial increase, followed by a decrease at 28 days after injury. These changes correlated with the band intensity ratio and the changes in distribution of axon and myelin in Raman spectral analysis. Thus, our results suggest that label-free biochemical imaging with Raman spectroscopy can be used to detect turnover of axon and myelin in peripheral nerve regeneration.

The mode of operation and theoretical concept behind a type of near-infrared spectrometer is discussed, which is used to measure concentrations of glucose, ethanol, CaCl ₂ , and KCl solutions in water, respectively. The main features of the instrument are its potential for short time-to-measurement resolution on the order of tens of milliseconds, its broad spectral bandwidth from 1.0 to 2.4 μm, and its ruggedness. These features allow the device to operate remotely in field applications and to utilize a wide variety of optical interfaces based on state-of-the-art fiber optic technology. Also, they provide a straightforward path to miniaturization with the concomitant enhancement in time resolution and applicability of the instrument and the technique.

High-throughput partial wave spectroscopy (HTPWS) is introduced as a high-speed spectral nanocytology technique that utilizes the field effect of carcinogenesis to perform minimally invasive cancer screening on at-risk populations. HTPWS uses fully automated hardware and an acousto-optic tunable filter to scan slides at low magnification, to select cells, and to rapidly acquire spectra at each spatial pixel in a cell between 450 and 700 nm, completing measurements of 30 cells in 40 min. Statistical quantitative analysis on the size and density of intracellular nanostructures extracted from the spectra at each pixel in a cell yields the diagnostic biomarker, disorder strength (L _d ). Linear correlation between L _d and the length scale of nanostructures was measured in phantoms with R ² =0.93 . Diagnostic sensitivity was demonstrated by measuring significantly higher L _d from a human colon cancer cell line (HT29 control vector) than a less aggressive variant (epidermal growth factor receptor knockdown). Clinical diagnostic performance for lung cancer screening was tested on 23 patients, yielding a significant difference in L _d between smokers and cancer patients, p=0.02 and effect size=1.00 . The high-throughput performance, nanoscale sensitivity, and diagnostic sensitivity make HTPWS a potentially clinically relevant modality for risk stratification of the large populations at risk of developing cancer.

We report the development of photoacoustic flowmetry assisted by high-intensity focused ultrasound (HIFU). This novel method employs HIFU to generate a heating impulse in the flow medium, followed by photoacoustic monitoring of the thermal decay process. Photoacoustic flowmetry in a continuous medium remains a challenge in the optical diffusive regime. Here, both the HIFU heating and photoacoustic detection can focus at depths beyond the optical diffusion limit (∼1  mm in soft tissue). This method can be applied to a continuous medium, i.e., a medium without discrete scatterers or absorbers resolvable by photoacoustic imaging. Flow speeds up to 41  mm⋅s ⁻¹ have been experimentally measured in a blood phantom covered by 1.5-mm-thick tissue.

We investigated the potential for using polydimethylsiloxane microfluidic devices in a biological assay to explore the cellular stress response (CSR) associated with hyperthermia induced by exposure to laser radiation. In vitro studies of laser-tissue interaction traditionally involved exposing a monolayer of cells. Given the heating-cooling dynamics of the cells and nutrient medium, this technique produces a characteristic “bulls-eye” temperature history that plagues downstream molecular analyses due to the nonuniform thermal experience of exposed cells. To circumvent this issue, we devised an approach to deliver single cells to the laser beam using a microfluidic channel, allowing homogeneous irradiation and collection of sufficient like-treated cells to measure changes in CSR after laser heating. To test this approach, we irradiated Jurkat-T cells with a 2-μm-wavelength laser in one branch of a 100-μm-wide bifurcated channel while unexposed control cells were simultaneously passing through the other, identical channel. Cell viability was measured using vital dyes, and expression of HSPA1A was measured using reverse transcription polymerase chain reaction. The laser damage threshold was 25±2  J/cm ² , and we found a twofold increase in expression at that exposure. This approach may be employed to examine transcriptome-wide/proteome changes and further comparative work across stressors and cell types.

We report the dynamic refractive index (RI) change of tissue under a stepped compression load using a custom-built pressure apparatus. Angle-dependent reflectance profiles of biotissue samples are recorded, and the RI values are resolved using the derivative total reflection method. These results are relevant for understanding the mechanism of mechanical optical clearing, for investigating tissue dynamics under mechanical stimuli, and for other biomedical applications.

Carotenoids are known to play an important role in health and disease state of living human tissue based on their antioxidant and optical filtering functions. In this study, we show that carotenoids exist in human bone and surrounding fatty tissue both in significant and individually variable concentrations. Measurements of biopsied tissue samples with molecule-specific Raman spectroscopy and high-performance liquid chromatography reveal that all carotenoids that are known to exist in human skin are also present in human bone. This includes all carotenes, lycopene, β -cryptoxanthin, lutein, and zeaxanthin. We propose quantitative reflection imaging as a noncontact optical method suitable for the measurement of composite carotenoid levels in bone and surrounding tissue exposed during open surgeries such as total knee arthroplasty, and as a proof of concept, demonstrate carotenoid measurements in biopsied bone samples. This will allow one to establish potential correlations between internal tissue carotenoid levels and levels in skin and to potentially use already existing optical skin carotenoid tests as surrogate marker for bone carotenoid status.

Functional near infrared spectroscopy (fNIRS) is a powerful tool for the study of oxygenation and hemodynamics of living tissues. Despite the continuous nature of the processes generating the data, analysis of fNIRS data has been limited to discrete-time methods. We propose a technique, namely functional data analysis (fDA), that converts discrete samples to continuous curves. We used fNIRS data collected on forehead during a cold pressor test (CPT) from 20 healthy subjects. Using functional principal component analysis, oxyhemoglobin (HbO₂) and deoxyhemoglobin (Hb) curves were decomposed into several components based on variability across the subjects. Each component corresponded to an experimental condition and provided qualitative and quantitative information of the shape and weight of that component. Furthermore, we applied functional canonical correlation analysis to investigate the interaction between Hb and HbO₂ curves. We showed that the variation of Hb and HbO₂ was positively correlated during the CPT, with a “far” channel on right forehead showing a smaller and faster HbO ₂ variation than Hb. This research suggests the fDA platform for the analysis of fNIRS data, which solves problem of high dimensionality, enables study of response dynamics, enhances characterization of the evoked response, and may improve design of future fNIRS experiments.

We demonstrate the first application of Raman spectroscopy in diagnosing nonmalignant, premalignant, malignant, and metastatic stages of breast cancer in a three-dimensional (3-D) cell culture model that closely mimics an in vivo environment. Comprehensive study comparing classification in two-dimensional (2-D) and 3-D cell models was performed using statistical methods composed of principal component analysis for exploratory analysis and outlier removal, partial least squares discriminant analysis, and elastic net regularized regression for classification. Our results show that Raman spectroscopy with an appropriate classification tool has excellent resolution to discriminate the four stages of breast cancer progression, with a near 100% accuracy for both 2-D and 3-D cell models. The diversity in chemical groups related to nucleic acids, proteins, and lipids, among other chemicals, were identified by appropriate peaks in the Raman spectra that correspond to the correct classification of the different stages of tumorigenesis model comprising of MCF10A, MCF10AneoT, MCF10CA1h, and MCF10CA1a cell lines. An explicit relationship between wavenumber and the stages of cancer progression was identified by the elastic net variable selection.

Application of the specular reflection intensity was previously reported for the quantification of early dental erosion. Further development of the technique and assembly of the miniaturized pen-size instrument are described. The optical system was adjusted to fit into a handy device which could potentially access different positions in the oral cavity. The assembled instrument could successfully detect early erosion progression in both polished (n=70 ) and native (n=20 ) human enamels. Different severities of enamel erosion were induced by varying incubation time of polished enamel in 1% citric acid (pH=3.60 , 0.5 to 10 min), while the native incisors were treated in the commercial orange juice (Tropicana Pure Premium®, pH=3.85 , 10 to 60 min). The instrument provided a good differentiation between various severities of the erosion in vitro. The size of the measurement spot affected the erosion monitoring in native enamel (human incisors). The erosion measurement in the 0.7-mm (diameter) cervical spots showed systematically lower reflection intensities compared with the analysis of central and incisal small spots. The application of larger spot areas (2.3 mm) for the erosion monitoring revealed no effect (p<0.05 ) of the spot position on the reflection signal. High variation of the teeth susceptibility toward in vitro erosion was detected in native enamel.

Optical properties of fresh and frozen tissues of rat heart, kidney, brain, liver, and muscle were measured in the 450- to 700-nm range. The total reflectance and transmittance were measured using a well-calibrated integral sphere set-up. Absorption coefficient μ_a and reduced scattering coefficient μ′_s were derived from the experimental measurements using the inverse adding doubling technique. The influence of cryogenic processing on optical properties was studied. Interindividual and intraindividual variations were assessed. These new data aim at filling the lack of validated optical properties in the visible range especially in the blue-green region of particular interest for fluorescence and optogenetics preclinical studies. Furthermore, we provide a unique comparison of the optical properties of different organs obtained using the same measurement set-up for fresh and frozen tissues as well as an estimate of the intraindividual and interindividual variability.

Toward developing a new method for restoring tissue viscoelasticity in scarred vocal folds, we previously proposed a method to localize biomaterials within subepithelial voids ablated using ultrafast laser pulses. The clinical implementation of this method necessitates the quantification of the laser parameters for ablating scarred tissue. Here, we present a comprehensive study of these parameters including ablation threshold and bubble lifetime in healthy and scarred tissues. We also present a new method for extracting tissue-specific ablation threshold and scattering lengths of different tissue layers. This method involves finding the ablation threshold at multiple depths and solving the equations based on Beer’s law of light attenuation for each depth to estimate the unknown parameters. Measured threshold fluences were 1.75  J/cm ² for vocal folds and 0.5  J/cm ² for cheek pouches for 3-ps, 776-nm laser pulses. Scarred pouches exhibited 30% lower threshold than healthy pouches, possibly due to the degraded mechanical properties of scarred collagen during wound healing. The analysis of tissue architecture indicated a direct correlation between the ablation threshold and tissue tensile strength and that the bubble lifetime is inversely related to tissue stiffness. Overall, this study sheds light on the required laser parameters for successful implementation of ultrafast laser ablation for phonosurgery.

We have presented an innovative laser-assisted reduction surgery (LARS) based on plasma-induced ablation and photodisruption effects. In addition, we developed a laser operation system. Fetuses of mice from the Institute for Cancer Research that were immersed in physiological saline were irradiated by convergent-pulsed laser with a wavelength of 1064 nm, pulse width of 6 ns, and pulse energy of 50 mJ. The hearts of the postirradiated fetuses were significantly damaged, which resulted in rapid fetal death. We also substantiated the safety of LARS by analyzing the heat distribution of the induced laser pulse with thermal distribution equations. The results demonstrate that this innovative method for pregnancy reduction is feasible.

Alzheimer’s disease irreversibly and progressively damages the brain, but the treatments in clinical trials are too slow. So, we hypothesized that the presence of erythrocyte variants with AD could be used as a noninvasive means to predict or trigger for administration of the preventive therapeutics, and the aim of this study is to develop a method using Raman spectroscopy in a rat model of Aβ₂₅₋₃₅ -induced neurotoxicity, and then evaluate the protective effect of bajijiasu by this method. Results showed that the Raman spectra fingerprints of the erythrocyte of model group were obvious different from those of the normal control, as peaks around the region 650  ^cm−1 belonged to the s–s makers, 1605  cm⁻¹ corresponded to the high spin (deoxygenated-Hb) marker, 1374  cm⁻¹ arises from ν4 as a sign of concentration of O₂ , and 1123 and 1033  cm⁻¹ are associated with the trans stretching vibrations of CAC skeleton. Results also showed that bajijiasu can make these changes recover. Our study also suggested that erythrocyte variants detected using Raman spectroscopy should be tested in a specific longitudinal study for the association with AD diagnosis, and if positive, can be used as a prognostic marker.