TOPICS: In vivo imaging, Diffuse reflectance spectroscopy, Skin, Spectroscopy, Tissue optics, Tissues, Control systems, Near infrared, Optical properties, Calibration
Contact pressure induced by manually operated fiber optic probes can significantly affect the optical properties of the studied tissue. If the contact pressure and the changes in optical properties are measured properly, then the complementary information can be used to obtain additional insight into the tissue physiology. However, as reliable assessment of the contact pressure in the existing diffuse reflectance setups is difficult, the impact of contact pressure is usually neglected or considered as a source of errors. We introduce a measurement system for controlled application of contact pressure and for the acquisition of diffuse reflectance spectra, which is suitable for in vivo studies and for overcoming the limitations of the existing measurement setups. A spectral-contact-pressure plane is proposed to present the combined information, highlighting the unique tissue response to the applied pressure.
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The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease.
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Special Section on Optical Coherence Tomography and Interferometry: Advanced Engineering and Biomedical Applications
Since its invention in the late ‘80s and early ‘90s, optical coherence interferometry (OCI) and its imaging version, optical coherence tomography (OCT), techniques experienced rapid scientific and technological advancements allowing high-resolution imaging and analysis of tissues and cells in three dimensions, with micrometer-level resolution and with speeds approaching and recently exceeding video rate. The unique capabilities of OCI and OCT to assess tissues, coupled with their noninvasive and contrast agent-free nature has resulted in a wide variety of exciting biomedical applications across the clinical spectrum, including ophthalmology, cardiology, and dentistry, among others. As a result, many labs and more than 40 OCT start-up companies currently invest significant resources for new OCT technology and product developments, for both preclinical and clinical use.
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We present a three-dimensional (3-D) computational method to detect soft tissue sarcomas with the goal of automatic surgical margin assessment based on optical coherence tomography (OCT) images. Three parameters are investigated and quantified from OCT images as the indicators for the tissue diagnosis including the signal attenuation (A-line slope), the standard deviation of the signal fluctuations (speckles), and the exponential decay coefficient of its spatial frequency spectrum. The detection of soft tissue sarcomas relies on the combination of these three parameters, which are related to the optical attenuation characteristics and the structural features of the tissue. Pilot experiments were performed on ex vivo human tissue samples with homogeneous pieces (both normal and abnormal) and tumor margins. Our results demonstrate the feasibility of this computational method in the differentiation of soft tissue sarcomas from normal tissues. The features of A-line-based detection and 3-D quantitative analysis yield promise for a computer-aided technique capable of accurately and automatically identifying resection margins of soft tissue sarcomas during surgical treatment.
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Microcirculation imaging is a key parameter for studying the pathophysiological processes of various disease conditions, in both clinical and fundamental research. A full-range spectral-domain correlation mapping optical coherence tomography (cm-OCT) method to obtain a complex-conjugate-free, full-range depth-resolved microcirculation map is presented. The proposed system is based on a high-speed spectrometer at 91 kHz with a modified scanning protocol to achieve higher acquisition speed to render cm-OCT images with high-speed and wide scan range. The mirror image elimination is based on linear phase modulation of B-frames by introducing a slight off-set of the probe beam with respect to the lateral scanning fast mirror’s pivot axis. An algorithm that exploits the Hilbert transform to obtain a complex-conjugate-free image in conjunction with the cm-OCT algorithm is used to obtain full-range imaging of microcirculation within tissue beds in vivo. The estimated sensitivity of the system was around 105 dB near the zero-delay line with ∼20 dB roll-off from ±0.5 to ±3 mm imaging-depth position. The estimated axial and lateral resolutions are ∼12 and ∼30 μm , respectively. A direct consequence of this complex conjugate artifact elimination is the enhanced flow imaging sensitivity for deep tissue imaging application by imaging through the most sensitive zero-delay line and doubling the imaging range.
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Intravascular optical coherence tomography (IVOCT) is rapidly becoming the method of choice for assessing vessel healing after stent implantation due to its unique axial resolution <20 μm . The amount of neointimal coverage is an important parameter. In addition, the characterization of neointimal tissue maturity is also of importance for an accurate analysis, especially in the case of drug-eluting and bioresorbable stent devices. Previous studies indicated that well-organized mature neointimal tissue appears as a high-intensity, smooth, and homogeneous region in IVOCT images, while lower-intensity signal areas might correspond to immature tissue mainly composed of acellular material. A new method for automatic neointimal tissue characterization, based on statistical texture analysis and a supervised classification technique, is presented. Algorithm training and validation were obtained through the use of 53 IVOCT images supported by histology data from atherosclerotic New Zealand White rabbits. A pixel-wise classification accuracy of 87% and a two-dimensional region–based analysis accuracy of 92% (with sensitivity and specificity of 91% and 93%, respectively) were found, suggesting that a reliable automatic characterization of neointimal tissue was achieved. This may potentially expand the clinical value of IVOCT in assessing the completeness of stent healing and speed up the current analysis methodologies (which are, due to their time- and energy-consuming character, not suitable for application in large clinical trials and clinical practice), potentially allowing for a wider use of IVOCT technology.
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We demonstrate intensity-based optical coherence tomography (OCT) angiography using the squared difference of two sequential frames with bulk-tissue-motion (BTM) correction. This motion correction was performed by minimization of the sum of the pixel values using axial- and lateral-pixel–shifted structural OCT images. We extract the BTM-corrected image from a total of 25 calculated OCT angiographic images. Image processing was accelerated by a graphics processing unit (GPU) with many stream processors to optimize the parallel processing procedure. The GPU processing rate was faster than that of a line scan camera (46.9 kHz). Our OCT system provides the means of displaying structural OCT images and BTM-corrected OCT angiographic images in real time.
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Optical coherence tomography (OCT) is a rapidly growing imaging modality, particularly in the field of ophthalmology. Accurate early diagnosis of diseases requires consistent and validated imaging performance. In contrast to more well-established medical imaging modalities, no standardized test methods currently exist for OCT quality assurance. We developed a retinal phantom which mimics the thickness and near-infrared optical properties of each anatomical retinal layer as well as the surface topography of the foveal pit. The fabrication process involves layer-by-layer spin coating of nanoparticle-embedded silicone films followed by laser micro-etching to modify the surface topography. The thickness of each layer and dimensions of the foveal pit are measured with high precision. The phantom is embedded into a commercially available, water-filled model eye to simulate ocular dispersion and emmetropic refraction, and for ease of use with clinical OCT systems. The phantom was imaged with research and clinical OCT systems to assess image quality and software accuracy. Our results indicate that this phantom may serve as a useful tool to evaluate and standardize OCT performance.
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An approach to elastographic mapping in optical coherence tomography (OCT) using comparison of correlation stability of sequentially obtained intensity OCT images of the studied strained tissue is discussed. The basic idea is that for stiffer regions, the OCT image is distorted to a smaller degree. Consequently, cross-correlation maps obtained with compensation of trivial translational motion of the image parts using a sliding correlation window can represent the spatial distribution of the relative tissue stiffness. An important advantage of the proposed approach is that it allows one to avoid the stage of local-strain reconstruction via error-sensitive numerical differentiation of experimentally determined displacements. Another advantage is that the correlation stability (CS) approach intrinsically implies that for deformed softer tissue regions, cross-correlation should already be strongly decreased in contrast to the approaches based on initial reconstruction of displacements. This feature determines a much wider strain range of operability than the proposed approach and is favorable for its free-hand implementation using the OCT probe itself to deform the tissue. The CS approach can be implemented using either the image elements reflecting morphological structure of the tissue or performing the speckle-level cross-correlation. Examples of numerical simulations and experimental demonstrations using both phantom samples and in vivo obtained OCT images are presented.
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A fast swept-source optical coherence tomography (SS-OCT) system is employed to acquire volumes of dental tissue, in order to monitor the temporal evolution of dental wear. An imaging method is developed to evaluate the volume of tissue lost in ex vivo artificially induced abfractions and attritions. The minimal volume (measured in air) that our system could measure is 2352 μm 3 . A volume of 25,000 A-scans is collected in 2.5 s. All these recommend the SS-OCT method as a valuable tool for dynamic evaluation of the abfraction and attrition with remarkable potential for clinical use.
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A comparative study of physical, chemical, and combined enhancement of transdermal transport of optical clearing agents (OCAs) is presented. As a physical enhancer of diffusivity, ultrasound (US) with a frequency 1 MHz and a power 1.1 W in the continuous mode was used, and dimethyl sulfoxide (DMSO) was used as a chemical enhancer. OCA (glycerol and polyethylene glycol-400 in equal proportion) was topically applied to the rat skin in vivo as alone or as together with the enhancers. Monitoring of skin optical clearing was implemented using an optical coherence tomography. The results have shown that the attenuation coefficient of intact skin dermis after the application of US-DMSO-OCA, US-OCA (both for 4 min), and DMSO-OCA (for 20 min) combinations decreased approximately by 31%, 19%, and 5%, respectively, while OCA alone did not induce a noticeable clearing effect for 20 min. Control skin sites with removed epidermis were used for modeling the upper limit of dermis optical clearing, i.e., maximal degree of optical clearing, by using the studied enhancers. They demonstrated that the attenuation coefficient decreases by 32%, 30%, 17%, and 16% at the action of US-DMSO-OCA, US-OCA, DMSO-OCA, and OCA, respectively. It can be concluded that US-DMSO-OCA combination only allowed reaching the upper limit of skin optical clearing.
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TOPICS: Optical coherence tomography, Fiber optics, Polarization, Phase modulation, Calibration, Mirrors, In vivo imaging, Birefringence, Signal to noise ratio, Interferometers
A swept source (SS)-based circular-state (CS) polarization-sensitive optical coherence tomography (PS-OCT) constructed entirely with polarization-maintaining fiber optics components is proposed with the experimental verification. By means of the proposed calibration scheme, bulk quarter-wave plates can be replaced by fiber optics polarization controllers to, therefore, realize an all-fiber optics CS SSPS-OCT. We also present a numerical dispersion compensation method, which can not only enhance the axial resolution, but also improve the signal-to-noise ratio of the images. We demonstrate that this compact and portable CS SSPS-OCT system with an accuracy comparable to bulk optics systems requires less stringent lens alignment and can possibly serve as a technology to realize PS-OCT instrument for clinical applications (e.g., endoscopy). The largest deviations in the phase retardation (PR) and fast-axis (FA) angle due to sample probe in the linear scanning and a rotation angle smaller than 65 deg were of the same order as those in stationary probe setups. The influence of fiber bending on the measured PR and FA is also investigated. The largest deviations of the PR were 3.5 deg and the measured FA change by ∼12 to 21 deg. Finally, in vivo imaging of the human fingertip and nail was successfully demonstrated with a linear scanning probe.
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The formation of burn-scar tissue in human skin profoundly alters, among other things, the structure of the dermis. We present a method to characterize dermal scar tissue by the measurement of the near-infrared attenuation coefficient using optical coherence tomography (OCT). To generate accurate en face parametric images of attenuation, we found it critical to first identify (using speckle decorrelation) and mask the tissue vasculature from the three-dimensional OCT data. The resulting attenuation coefficients in the vasculature-masked regions of the dermis of human burn-scar patients are lower in hypertrophic (3.8±0.4 mm −1 ) and normotrophic (4.2±0.9 mm −1 ) scars than in contralateral or adjacent normal skin (6.3±0.5 mm −1 ). Our results suggest that the attenuation coefficient of vasculature-masked tissue could be used as an objective means to assess human burn scars.
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The overexpression of human epidermal growth factor receptor 2 (HER2) is associated with increased breast cancer recurrence and worse prognosis. Effective treatments such as trastuzumab and lapatinib for patients with HER2 overexpression target the blockade of HER2 signaling activities but are often limited by the emergence of acquired drug resistance. This study applied Raman spectroscopy to differentially identify the amplification status of HER2 in cells and to characterize the biochemical composition of lapatinib resistant and sensitive HER2+ breast cancer cells in response to the drug. Raman spectra from BT474 (HER2+ breast cancer cell), MCF-10A (HER2− control), and HER2+ MCF-10A (HER2+ control) were analyzed using lasso and elastic-net regularized generalized linear models (glmnet) for multivariate statistical analysis and were discriminated to groups of different HER2 expression status with an overall 99% sensitivity and specificity. Enhanced lipid content and decreased proteome were observed in HER2+ cells. With lapatinib treatment, lapatinib-resistant breast cancer cells demonstrated sustained lipogenesis compared with the sensitive cells.
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We evaluated the potential of the Cramér–Rao lower bound (CRLB) to serve as a design metric for diffuse optical imaging systems. The CRLB defines the best achievable precision of any estimator for a given data model; it is often used in the statistical signal processing community for feasibility studies and system design. Computing the CRLB requires inverting the Fisher information matrix (FIM), however, which is usually ill-conditioned (and often underdetermined) in the case of diffuse optical tomography (DOT). We regularized the FIM by assuming that the inhomogeneity to be imaged was a point target and assessed the ability of point-target CRLBs to predict system performance in a typical DOT setting in silico. Our reconstructions, obtained with a common iterative algebraic technique, revealed that these bounds are not good predictors of imaging performance across different system configurations, even in a relative sense. This study demonstrates that agreement between the trends predicted by the CRLBs and imaging performance obtained with reconstruction algorithms that rely on a different regularization approach cannot be assumed a priori. Moreover, it underscores the importance of taking into account the intended regularization method when attempting to optimize source–detector configurations.
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Callus formation is a critical step for successful fracture healing. Little is known about the molecular composition and mineral structure of the newly formed tissue in the callus. The aim was to evaluate the feasibility of small angle x-ray scattering (SAXS) to assess mineral structure of callus and cortical bone and if it could provide complementary information with the compositional analyses from Fourier transform infrared (FTIR) microspectroscopy. Femurs of 12 male Sprague–Dawley rats at 9 weeks of age were fractured and fixed with an intramedullary 1.1 mm K-wire. Fractures were treated with the combinations of bone morphogenetic protein-7 and/or zoledronate. Rats were sacrificed after 6 weeks and both femurs were prepared for FTIR and SAXS analysis. Significant differences were found in the molecular composition and mineral structure between the fracture callus, fracture cortex, and control cortex. The degree of mineralization, collagen maturity, and degree of orientation of the mineral plates were lower in the callus tissue than in the cortices. The results indicate the feasibility of SAXS in the investigation of mineral structure of bone fracture callus and provide complementary information with the composition analyzed with FTIR. Moreover, this study contributes to the limited FTIR and SAXS data in the field.
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The quantification of skin carotenoid levels has a range of applications in Caucasian populations, from serving as a versatile and noninvasive biomarker (e.g., of systemic carotenoid levels, carotenoid consumption, the antioxidative capacity of skin, and oxidative stress) to being used in appearance-based interventions. Yet, no study has investigated the quantitative effect of carotenoid supplementation on African skin. The aim of this study was to determine if beta-carotene supplementation produces a significant color change in three different regions of African skin. To do so we supplemented the diet of African participants with beta-carotene over an eight-week period. Reflectance spectrophotometry measurements were taken on a weekly basis for the duration of the supplementation study. Results show a significant increase in the carotenoid coloration of lightly pigmented skin (palm of the hand) and highly pigmented skin with low sun exposure (inner arm) after supplementation. The latter was no longer significant after Bonferroni correction. The carotenoid coloration of highly pigmented skin areas with high sun exposure did not increase significantly. Skin carotenoid measurements of the palm of the hand might, therefore, serve as a potential biomarker for systemic carotenoid concentrations in people of African descent.
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Within the framework of further development of unified Monte Carlo code for the needs of biomedical optics and biophotonics, we present an approach for modeling of coherent polarized light propagation in highly scattering turbid media, such as biological tissues. The temporal coherence of light, linear and circular polarization, interference, and the helicity flip of circularly polarized light due to reflection at the medium boundary and/or backscattering events are taken into account. To achieve higher accuracy in the results and to speed up the modeling, the implementation of the code utilizes parallel computing on NVIDIA graphics processing units using Compute Unified Device Architecture. The results of the simulation of coherent linearly and circularly polarized light are presented in comparison with the results of known theoretical studies and the results of alternative modelings.
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In this report, we describe a graphics processing unit (GPU)-accelerated processing platform for real-time acquisition and display of flow contrast images with Fourier domain optical coherence tomography (FDOCT) in mouse and human eyes in vivo. Motion contrast from blood flow is processed using the speckle variance OCT (svOCT) technique, which relies on the acquisition of multiple B-scan frames at the same location and tracking the change of the speckle pattern. Real-time mouse and human retinal imaging using two different custom-built OCT systems with processing and display performed on GPU are presented with an in-depth analysis of performance metrics. The display output included structural OCT data, en face projections of the intensity data, and the svOCT en face projections of retinal microvasculature; these results compare projections with and without speckle variance in the different retinal layers to reveal significant contrast improvements. As a demonstration, videos of real-time svOCT for in vivo human and mouse retinal imaging are included in our results. The capability of performing real-time svOCT imaging of the retinal vasculature may be a useful tool in a clinical environment for monitoring disease-related pathological changes in the microcirculation such as diabetic retinopathy.
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A method for measuring three-dimensional (3-D) direction images of collagen fibers in biological tissue is presented. Images of the 3-D directions are derived from the measured transmission Mueller matrix images (MMIs), acquired at different incidence angles, by taking advantage of the form birefringence of the collagen fibers. The MMIs are decomposed using the recently developed differential decomposition, which is more suited to biological tissue samples than the common polar decomposition method. Validation of the 3-D direction images was performed by comparing them with images from second-harmonic generation microscopy. The comparison found a good agreement between the two methods. It is envisaged that 3-D directional imaging could become a useful tool for understanding the collagen framework for fibers smaller than the diffraction limit.
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Photoacoustic (PA) techniques can measure temperature in biological tissues because PA signal amplitude is sensitive to tissue temperature. So far, temperature-measuring PA techniques have focused on sensing of temperature changes at a single position. In this work, we photoacoustically measured spatial distribution of temperature in deep tissue. By monitoring the temperature at a single position using a thermocouple, the relationship between the PA signal amplitude and the actual temperature was determined. The relationship was then used to translate a PA image into a temperature map. This study showed that it is possible to calibrate the system for the temperature range of hyperthermia using single-point measurements over a smaller temperature range. Our experimental results showed a precision of −0.8±0.4°C (mean±standard error ) in temperature measurement, and a spatial resolution as fine as 1.0 mm. PA techniques can be potentially applied to monitor temperature distribution deep in tissue during hyperthermia treatment of cancer.
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The Geant4 Application for Emission Tomography (GATE) is an advanced open-source software dedicated to Monte-Carlo (MC) simulations in medical imaging involving photon transportation (Positron emission tomography, single photon emission computed tomography, computed tomography) and in particle therapy. In this work, we extend the GATE to support simulations of optical imaging, such as bioluminescence or fluorescence imaging, and validate it against the MC for multilayered media standard simulation tool for biomedical optics in simple geometries. A full simulation set-up for molecular optical imaging (bioluminescence and fluorescence) is implemented in GATE, and images of the light distribution emitted from a phantom demonstrate the relevance of using GATE for optical imaging simulations.
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We present a pilot clinical application of coherent hemodynamics spectroscopy (CHS), a technique to investigate cerebral hemodynamics at the microcirculatory level. CHS relies on frequency-resolved measurements of induced cerebral hemodynamic oscillations that are measured with near-infrared spectroscopy (NIRS) and analyzed with a hemodynamic model. We have used cyclic inflation (200 mmHg) and deflation of a pneumatic cuff placed around the subject’s thigh at seven frequencies in the range of 0.03 to 0.17 Hz to generate CHS spectra and to obtain a set of physiological parameters that include the blood transit times in the cerebral microcirculation, the cutoff frequency for cerebral autoregulation, and blood volume ratios across the three different compartments. We have investigated five hemodialysis patients, during the hemodialysis procedure, and six healthy subjects. We have found that the blood transit time in the cerebral microcirculation is significantly longer in hemodialysis patients with respect to healthy subjects. No significant differences were observed between the two groups in terms of autoregulation efficiency and blood volume ratios. The demonstration of the applicability of CHS in a clinical setting and its sensitivity to the highly important cerebral microcirculation may open up new opportunities for NIRS applications in research and in medical diagnostics and monitoring.
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The natural history of atherosclerosis is marked by changes in the lipid biochemistry in the diseased arterial wall. As lesions become more vulnerable, different cholesterol species accumulate in the plaque. Understanding unstable atherosclerosis as a pharmacological and interventional therapeutic target requires chemically specific imaging of disease foci. In this study, we aim to image atherosclerotic plaque lipids and other vessel wall constituents with spectroscopic intravascular photoacoustics (sIVPA). sIVPA imaging can identify lipids in human coronary atherosclerotic plaque by relying on contrast in the near-infrared absorption spectra of the arterial wall components. Using reference spectra acquired on pure compounds, we analyzed sIVPA data from human coronary plaques ex vivo, to image plaque composition in terms of cholesterol and cholesterol ester content. In addition, we visualized the deeper lying connective tissue layers of the adventitia, as well as the fatty acid containing adipose cells in the peri-adventitial tissue. We performed simultaneous coregistered IVUS imaging to obtain complementary morphological information. Results were corroborated by histopathology. sIVPA imaging can distinguish the most prevalent lipid components of human atherosclerotic plaques and also visualize the connective tissue layers of the adventitia and the fatty acid containing adipose cells in the peri-adventitial tissue.
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Most methods for measuring light-tissue interaction focus on volume reflectance, while very few measure light transmission. In a previous work, we suggested investigating the influence of blood vessel diameter on photons exiting the tissue at all exit angles to receive the full scattering profile. By this method, we have shown that there is a central angle, i.e., the isobaric point, independent of blood vessel diameter. The vessel diameter changes the effective reduced scattering coefficient. However, both the scattering profile and the value of the isobaric point strongly depend on optical properties and the exact geometry of the tissue. In this study, we investigate the dependency of the isobaric point on tissue diameter and scattering coefficient in both two-dimensional and three-dimensional simulations. We show that the value of this point linearly depends on tissue diameter. The findings of this work solve the dilemma of whether to measure transmission or reflection since the isobaric point reduces by half the total amount of exiting photons. Furthermore, the full scattering profile is sensitive to changes in the scattering properties, but a single isobaric point to these changes is expected. If this point is not found, it is a diagnostic indication of an unexpected change in the tissue.
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TOPICS: 3D modeling, Tissue optics, Data modeling, Absorption, Reconstruction algorithms, Finite element methods, Hemodynamics, Brain, Head, 3D image processing
This paper proposes a new reconstruction method for diffuse optical tomography using reduced-order models of light transport in tissue. The models, which directly map optical tissue parameters to optical flux measurements at the detector locations, are derived based on data generated by numerical simulation of a reference model. The reconstruction algorithm based on the reduced-order models is a few orders of magnitude faster than the one based on a finite element approximation on a fine mesh incorporating a priori anatomical information acquired by magnetic resonance imaging. We demonstrate the accuracy and speed of the approach using a phantom experiment and through numerical simulation of brain activation in a rat’s head. The applicability of the approach for real-time monitoring of brain hemodynamics is demonstrated through a hypercapnic experiment. We show that our results agree with the expected physiological changes and with results of a similar experimental study. However, by using our approach, a three-dimensional tomographic reconstruction can be performed in ∼3 s per time point instead of the 1 to 2 h it takes when using the conventional finite element modeling approach.
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Optical coherence tomography (OCT) is a light-based intracoronary imaging modality that provides high-resolution cross-sectional images of the luminal and plaque morphology. Currently, the segmentation of OCT images and identification of the composition of plaque are mainly performed manually by expert observers. However, this process is laborious and time consuming and its accuracy relies on the expertise of the observer. To address these limitations, we present a methodology that is able to process the OCT data in a fully automated fashion. The proposed methodology is able to detect the lumen borders in the OCT frames, identify the plaque region, and detect four tissue types: calcium (CA), lipid tissue (LT), fibrous tissue (FT), and mixed tissue (MT). The efficiency of the developed methodology was evaluated using annotations from 27 OCT pullbacks acquired from 22 patients. High Pearson’s correlation coefficients were obtained between the output of the developed methodology and the manual annotations (from 0.96 to 0.99), while no significant bias with good limits of agreement was shown in the Bland-Altman analysis. The overlapping areas ratio between experts’ annotations and methodology in detecting CA, LT, FT, and MT was 0.81, 0.71, 0.87, and 0.81, respectively.
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Functional near-infrared spectroscopy (fNIRS), acquired simultaneously with electroencephalography (EEG), allows the investigation of hemodynamic brain responses to epileptic activity. Because the presumed epileptogenic focus is patient-specific, an appropriate source/detector (SD) montage has to be reconfigured for each patient. The combination of EEG and fNIRS, however, entails several constraints on montages, and finding an optimal arrangement of optodes on the cap is an important issue. We present a method for computing an optimal SD montage on an EEG/fNIRS cap that focuses on one or several specific brain regions; the montage maximizes the spatial sensitivity. We formulate this optimization problem as a linear integer programming problem. The method was evaluated on two EEG/fNIRS caps. We simulated absorbers at different locations on a head model and generated realistic optical density maps on the scalp. We found that the maps of optimal SD montages had spatial resolution properties comparable to those of regular SD arrangements for the whole head with significantly fewer sensors than regular SD arrangements. In addition, we observed that optimal montages yielded improved spatial density of fNIRS measurements over the targeted regions together with an increase in signal-to-noise ratio.
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Accurate segmentation of structural magnetic resonance images is critical for creating subject-specific forward models for functional neuroimaging source localization. In this work, we present an innovative segmentation algorithm that generates accurate head tissue layer thicknesses that are needed for diffuse optical tomography (DOT) data analysis. The presented algorithm is compared against other publicly available head segmentation methods. The proposed algorithm has a root mean square scalp thickness error of 1.60 mm, skull thickness error of 1.96 mm, and summed scalp and skull error of 1.49 mm. We also introduce a segmentation evaluation metric that evaluates the accuracy of tissue layer thicknesses in regions of the head where optodes are typically placed. The presented segmentation algorithm and evaluation metric are tools for improving the localization accuracy of neuroimaging with DOT, and also multimodal neuroimaging such as combined electroencephalography and DOT.
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Breast mimicking tissue optical phantoms with sufficient structural integrity to be deployed as stand-alone imaging targets are developed and successfully constructed with biologically relevant concentrations of water, lipid, and blood. The results show excellent material homogeneity and reproducibility with inter- and intraphantom variability of 3.5 and 3.8%, respectively, for water and lipid concentrations ranging from 15 to 85%. The phantoms were long-lasting and exhibited water and lipid fractions that were consistent to within 5% of their original content when measured 2 weeks after creation. A breast-shaped three-compartment model of adipose, fibroglandular, and malignant tissues was created with water content ranging from 30% for the adipose simulant to 80% for the tumor. Mean measured water content ranged from 30% in simulated adipose to 73% in simulated tumor with the higher water localized to the tumor-like material. This novel heterogeneous phantom design is composed of physiologically relevant concentrations of the major optical absorbers in the breast in the near-infrared wavelengths that should significantly improve imaging system characterization and optimization because the materials have stand-alone structural integrity and can be readily molded into the sizes and shapes of tissues commensurate with clinical breast imaging.
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Evaluation of spatiotemporal hemodynamic and metabolic responses during neural activation is crucial in studying brain function. We explore the use of a noninvasive multifunctional optical imaging system to measure these responses in a mouse brain upon electrically stimulated neural activation, with the cranium left intact. The system is developed by integrating an optical microangiography (OMAG) imaging system with a dual-wavelength laser speckle imaging (DW-LSI) system. The DW-LSI, running at an image acquisition speed of ∼100 Hz, is used to extract the large-scale two-dimensional map, revealing the localized response of blood flow, hemoglobin concentration, and metabolic rate of oxygen change. Guided by DW-LSI, the OMAG is, however, used to image the response of individual blood vessels with its unique depth-resolved capability. We show that the integrated system is capable of investigating neural activation, thus is potentially valuable in the preclinical study of the mechanism of neurovascular coupling.
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Photoacoustic section imaging reveals optically absorbing structures within a thin slice of an object. It requires measuring acoustic waves excited by absorption of short laser pulses with a cylindrical acoustic lens detector rotating around the object. Owing to the finite detector size and its limited depth of focus, various artifacts arise, seen as distortions within the imaging slice and cross-talk from neighboring areas of the object. The presented solution aims at avoiding these artifacts by a special design of the sensor and by use of a model-based reconstruction algorithm that improves section images by incorporating information from neighboring sections. The integrating property of the cylindrical detector, which exceeds in direction of the cylinder axis the size of the imaged object, avoids the lateral blurring that normally results from the finite width of a small detector. Applying a maximum likelihood reconstruction method for the inversion of the imaging system matrix to the temporal pressure signals yields line projections of the initial energy distribution, from which section images are obtained by applying the inverse Radon transform. By using data from few sections, a significant reduction of artifacts related to the imperfections of the sensor is demonstrated both in simulations and in phantom experiments.
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The challenge of depth-resolved, nonionizing (hybrid-optical) detection of mineral loss in bones is addressed using truncated-correlation photothermal coherence tomography (TC-PCT). This approach has importance not only in ground-based clinical procedures, but also in microgravity space applications. Analogous to x-ray morphometric parameters, two- and three-dimensional markers have been defined and estimated for chemically demineralized goat rib bones. Cortical and trabecular regions have been analyzed independently and together using the computational slicing advantage of TC-PCT, and the results have been verified using micro-CT imaging (the gold standard). For low-demineralization levels, both modalities follow the same trend. However, for very high mineral loss that is unlikely to occur naturally, anomalies exist in both methods. Demineralization tracking has been carried out to a depth of ∼3 mm below the irradiated surface. Compared with micro-CT imaging, TC-PCT offers an improved dynamic range, which is a beneficial feature while analyzing highly demineralized bones. Also, TC-PCT parameters are found to be more sensitive to trabecular and combined cortical-trabecular demineralization compared with x-ray parameters. Axial and lateral resolutions in bone imaging for the current instrumental configuration are ∼25 and 100 μm, respectively.
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Evaluation of tear film is performed by an optical reflectometer system with alignment guided by a galvanometer scanner. The reflectometer system utilizes optical fibers to deliver illumination light to the tear film and collect the film reflectance as a function of wavelength. Film thickness is determined by best fitting the reflectance-wavelength curve. The spectral reflectance acquisition time is 15 ms, fast enough for detecting film thickness changes. Fast beam alignment of 1 s is achieved by the galvanometer scanner. The reflectometer was first used to evaluate artificial tear film on a model eye with and without a contact lens. The film thickness and thinning rate have been successfully quantified with the minimum measured thickness of about 0.3 μm. Tear films in human eyes, with and without a contact lens, have also been evaluated. A high-contrast spectral reflectance signal from the precontact lens tear film is clearly observed, and the thinning dynamics have been easily recorded from 3.69 to 1.31 μm with lipid layer thickness variation in the range of 41 to 67 nm. The accuracy of the measurement is better than ±0.58% of the film thickness at an estimated tear film refractive index error of ±0.001 . The fiber-based reflectometer system is compact and easy to handle.
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We present initial steps toward a new measurement device enabling high-precision, noncontact remote and repeatable monitoring of intraocular pressure (IOP)-based on an innovative measurement principle. Using only a camera and a laser source, the device measures IOP by tracking the secondary speckle pattern trajectories produced by the reflection of an illuminating laser beam from the iris or the sclera. The device was tested on rabbit eyes using two different methods to modify IOP: via an infusion bag and via mechanical pressure. In both cases, the eyes were stimulated with increasing and decreasing ramps of the IOP. As IOP variations changed the speckle distributions reflected back from the eye, data were recorded under various optical configurations to define and optimize the best experimental configuration for the IOP extraction. The association between the data provided by our proposed device and that resulting from controlled modification of the IOP was assessed, revealing high correlation (R2 =0.98 ) and sensitivity and providing a high-precision measurement (5% estimated error) for the best experimental configuration. Future steps will be directed toward applying the proposed measurement principle in clinical trials for monitoring IOP with human subjects.
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Articular cartilage (AC) is mainly composed of collagen, proteoglycans, chondrocytes, and water. These constituents are inhomogeneously distributed to provide unique biomechanical properties to the tissue. Characterization of the spatial distribution of these components in AC is important for understanding the function of the tissue and progress of osteoarthritis. Fourier transform infrared (FT-IR) absorption spectra exhibit detailed information about the biochemical composition of AC. However, highly specific FT-IR analysis for collagen and proteoglycans is challenging. In this study, a chemometric approach to predict the biochemical composition of AC from the FT-IR spectra was investigated. Partial least squares (PLS) regression was used to predict the proteoglycan content (n=32 ) and collagen content (n=28 ) of bovine cartilage samples from their average FT-IR spectra. The optimal variables for the PLS regression models were selected by using backward interval partial least squares and genetic algorithm. The linear correlation coefficients between the biochemical reference and predicted values of proteoglycan and collagen contents were r=0.923 (p<0.001 ) and [i]r=0.896 ([i]p<0.001 ), respectively. The results of the study show that variable selection algorithms can significantly improve the PLS regression models when the biochemical composition of AC is predicted.
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Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing colonies from 80 strains belonging to nine bacterial and one yeast species directly on solid culture medium after 24-h (macrocolonies) and 6-h (microcolonies) incubation. This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing. Correct identification rates measured for macrocolonies and microcolonies reached 94.1% and 91.5%, respectively, in a leave-one-strain-out cross-validation mode without any correction for possible medium interference. Large spectral differences were observed between macrocolonies and microcolonies, that were attributed to true biological differences. Our results, conducted on a very diversified panel of species and strains, were obtained by using simple and robust sample preparation and preprocessing procedures, while still confirming published results obtained by using more complex elaborated protocols. Instrumentation is simplified by the use of 532-nm laser excitation yielding a Raman signal in the visible range. It is, to our knowledge, the first side-by-side full classification study of microorganisms in the exponential and stationary phases confirming the excellent performance of Raman spectroscopy for early species-level identification of microorganisms directly from an agar culture.
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According to the valence asymmetry hypothesis, the left/right asymmetry of prefrontal cortex (PFC) activity is correlated with specific emotional responses to mental stress and personality traits. Here, we evaluated the relation between emotional state and asymmetry in PFC activity at rest by using near-infrared spectroscopy (NIRS). We measured spontaneous oscillation of oxyhemoglobin (oxy-Hb) concentrations in the bilateral PFC at rest in normal adults employing two-channel NIRS. In order to analyze left/right asymmetry of PFC activity at rest, we calculated the laterality index at rest (LIR) (see text). We investigated the correlation between the LIR and anxiety levels evaluated by the State-Trait Anxiety Inventory (STAI) test. We found that the right PFC was more active at rest than the left PFC, corresponding to a higher anxiety level measured by the STAI; that is, subjects with right-dominant activity at rest showed higher STAI scores, while those with left-dominant oxy-Hb changes at rest showed lower STAI scores. Aging had no significant effect on the relation. The present results obtained by NIRS are consistent with the valence asymmetry hypothesis. We emphasize NIRS may be a useful tool for objective assessment of anxiety levels.
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Diffuse reflectance spectra (DRS) of biological samples are commonly measured using an integrating sphere (IS). To account for the incident light spectrum, measurement begins by placing a highly reflective white standard against the IS sample opening and collecting the reflected light. After replacing the white standard with the test sample of interest, DRS of the latter is determined as the ratio of the two values at each involved wavelength. However, such a substitution may alter the fluence rate inside the IS. This leads to distortion of measured DRS, which is known as single-beam substitution error (SBSE). Barring the use of more complex experimental setups, the literature states that only approximate corrections of the SBSE are possible, e.g., by using look-up tables generated with calibrated low-reflectivity standards. We present a practical method for elimination of SBSE when using IS equipped with an additional reference port. Two additional measurements performed at this port enable a rigorous elimination of SBSE. Our experimental characterization of SBSE is replicated by theoretical derivation. This offers an alternative possibility of computational removal of SBSE based on advance characterization of a specific DRS setup. The influence of SBSE on quantitative analysis of DRS is illustrated in one application example.
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Raman spectroscopy based discrimination of cervical precancer and normal tissue has been shown previously in vivo with fiber probe based measurements of colposcopically selected sites. With a view to developing in vivo large area imaging, macro raster scans of native cervical cone biopsies with an average of 200 spectra per sample are implemented (n=16 ). The diagnostic performance is evaluated using histopathological mapping of the cervix surface. Different data reduction and classification methods (principal component analysis, wavelets, k-nearest neighbors, logistic regression, partial least squares discriminant analysis) are compared. Using bootstrapping to estimate confidence intervals for sensitivity and specificity, it is concluded that differences among different spectra classification procedures are not significant. The classification performance is evaluated depending on the tissue pathologies included in the analysis using the average performance of different classification procedures. The highest sensitivity (89%) and specificity (86%) is obtained for the discrimination of normal squamous epithelium and high-grade precancer. When other non-high-grade tissue sites, such as columnar epithelium, metaplasia, and inflammation, are included, the diagnostic performance decreases.
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Thalassemia (Thal), sickle cell anemia, and iron deficiency anemia (IDA) are the most common blood disorders in many parts of the world, particularly in developing countries like India and Bangladesh. The well-established diagnostic procedure for them is the complete blood count (CBC); however, there is substantial confusion in discrimination between Thal and IDA blood samples based on such CBC. We propose a new spectral technique for reliable classification between the above two anemias. This is based on the identification and quantification of a certain set of fluorescent metabolites found in the blood samples of patients of Thal and IDA.
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The need for patient-specific photodynamic therapy (PDT) in dermatologic and oncologic applications has triggered several studies that explore the utility of surrogate parameters as predictive reporters of treatment outcome. Although photosensitizer (PS) fluorescence, a widely used parameter, can be viewed as emission from several fluorescent states of the PS (e.g., minimally aggregated and monomeric), we suggest that singlet oxygen luminescence (SOL) indicates only the active PS component responsible for the PDT. Here, the ability of discrete PS fluorescence-based metrics (absolute and percent PS photobleaching and PS re-accumulation post-PDT) to predict the clinical phototoxic response (erythema) resulting from 5-aminolevulinic acid PDT was compared with discrete SOL (DSOL)-based metrics (DSOL counts pre-PDT and change in DSOL counts pre/post-PDT) in healthy human skin. Receiver operating characteristic curve (ROC) analyses demonstrated that absolute fluorescence photobleaching metric (AFPM) exhibited the highest area under the curve (AUC) of all tested parameters, including DSOL based metrics. The combination of dose-metrics did not yield better AUC than AFPM alone. Although sophisticated real-time SOL measurements may improve the clinical utility of SOL-based dosimetry, discrete PS fluorescence-based metrics are easy to implement, and our results suggest that AFPM may sufficiently predict the PDT outcomes and identify treatment nonresponders with high specificity in clinical contexts.
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Keratinocytes play a central role in wound healing by responding to tissue injury through the activation of cellular proliferation and migration. Current clinical evidence suggests that the laser phototherapy (LPT) accelerates wound healing in a variety of oral diseases; however, the molecular mechanisms involved in response to LPT are not fully understood. Oral keratinocytes (NOK-SI) maintained under nutritional-deficit culture medium (2% fetal bovine serum) were irradiated with InGaAlP laser (660 nm; 40 mW; 0.04 cm 2 spot size) in punctual and contact modes. The energy densities used were 4 and 20 J/cm 2 corresponding to 4 and 20 s of exposure times and 0.16 and 0.8 J of energy per point, respectively. Three sessions of irradiations were applied with 6-h intervals. Further, the impact of LPT over cellular migration, proliferation, and activation of the mammalian target of rapamycin (mTOR) pathway, known to play a major role in epithelial migration and wound healing, was analyzed. Compared with control cells, the LPT-treated cells showed accelerated cellular migration without any changes in proliferation. Furthermore, LPT resulted in an increase in the phospho-S6 ribosomal protein, indicating activation of the mTOR signaling pathway. Collectively, these findings suggest that the LPT activates mTOR signaling pathway, promotes epithelial cell migration, and accelerates healing of oral mucosa.
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Pulsed lasers emitting wavelengths near 400 nm can selectively ablate dental calculus without damaging underlying and surrounding sound dental hard tissue. Our results indicate that calculus ablation at this wavelength relies on the absorption of porphyrins endogenous to oral bacteria commonly found in calculus. Sub- and supragingival calculus on extracted human teeth, irradiated with 400-nm, 60-ns laser pulses at ≤8 J/cm 2 , exhibits a photobleached surface layer. Blue-light microscopy indicates this layer highly scatters 400-nm photons, whereas fluorescence spectroscopy indicates that bacterial porphyrins are permanently photobleached. A modified blow-off model for ablation is proposed that is based upon these observations and also reproduces our calculus ablation rates measured from laser profilometry. Tissue scattering and a stratified layering of absorbers within the calculus medium explain the gradual decrease in ablation rate from successive pulses. Depending on the calculus thickness, ablation stalling may occur at <5 J/cm 2 but has not been observed above this fluence.
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This article [J. Biomed. Opt.. 17, (6 ), 066018 (2012)] was originally published online on 6 June 2012 with errors Table 2 on p. 4. The corrected table is reprinted below.
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Reviewed by Alberto Diaspro, SPIE Fellow, Istituto Italiano di Tecnologia, Genova, ItalyResearch and development of modern optical microscopes are guided by several aspects, from resolution obsession to imaging of living specimens, from a low-level of perturbation to long-term experiments on biological systems. One of the most outstanding goals is the possibility of performing at the highest current levels, including 4-D (x,y,z,t) nanoscopy/superresolution, label-free imaging. Coherent Raman scattering (CRS) microscopy represents a key label-free imaging method for studying living cells and tissues at a high time rate. Ji-Xin Cheng and Xiaoliang Sunney Xie, two of the most representative scientists in this area, assembled the perfect book for disseminating the method. Their experience as scientists and authors and their robustness and consideration in the field allowed them to edit a great book that, starting from the foundation of CRS microscopy, covers the field in a comprehensive and exhaustive way.
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