Breast density is a recognized strong and independent risk factor for breast cancer. We propose the use of time-resolved transmittance spectroscopy to estimate breast tissue density and potentially provide even more direct information on breast cancer risk. Time-resolved optical mammography at seven wavelengths (635 to 1060 nm) is performed on 49 subjects. Average information on breast tissue of each subject is obtained on oxy- and deoxyhemoglobin, water, lipids, and collagen content, as well as scattering amplitude and power. All parameters, except for blood volume and oxygenation, correlate with mammographic breast density, even if not to the same extent. A synthetic optical index proves to be quite effective in separating different breast density categories. Finally, the estimate of collagen content as a more direct means for the assessment of breast cancer risk is discussed.
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Selective excitation of a particular fluorophore in the presence of others demands clever design of the optical field interacting with the molecules. We describe the use of 20- to 50-GHz pulse-train excitation leading to two-photon absorption, followed by successive one-photon stimulated emission as a potential technique in the context of controlling two-photon molecular fluorescence, with applications in microscopy.
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Given that bone is an intriguing nanostructured dielectric as a partially disordered complex structure, we apply an elastic light scattering-based approach to image prefailure deformation and damage of bovine cortical bone under mechanical testing. We demonstrate that our imaging method can capture nanoscale deformation in a relatively large area. The unique structure, the high anisotropic property of bone, and the system configuration further allow us to use the transfer matrix method to study possible spectroscopic manifestations of prefailure deformation. Our sensitive yet simple imaging method could potentially be used to detect nanoscale structural and mechanical alterations of hard tissue and biomaterials in a fairly large field of view.
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Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.
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TOPICS: Positron emission tomography, Imaging systems, Tumors, Optical imaging, In vitro testing, In vivo imaging, Tissue optics, Molecular imaging, Photons, Medical research
Reporter gene/reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene/reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene/nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine ([18F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene/reporter probe techniques in medical research.
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A miniature fiber optic endomicroscope with built-in dynamic focus scanning capability is developed for the first time for 3-D two-photon fluorescence (TPF) imaging of biological samples. Fast 2-D lateral beam scanning is realized by resonantly vibrating a double-clad fiber cantilever with a tubular piezoactuator. Slow axial scanning is achieved by moving the distal end of the imaging probe with an extremely compact electrically driven shape memory alloy (SMA). The 10-mm-long SMA allows 150-μm contractions with a driving voltage varying only from 50 to 100 mV. The response of the SMA contraction with the applied voltage is nonlinear, but repeatable and can be accurately calibrated. Depth-resolved imaging of acriflavine-stained biological tissues and unstained white paper with the endomicroscope is performed, and the results demonstrate the feasibility of 3-D nonlinear optical imaging with the SMA-based scanning fiber-optic endomicroscope.
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We present a simple and cost-effective optical technique for the simultaneous assessment of pulsating and total blood noninvasively in an inflammatory skin lesion. Acquisitions of diffuse reflectance spectra in the visible range at 6 Hz are used to trace the oscillating components of reflectance. Measurements on erythematous lesions from a UV insult show slow changing signal at about 0.1 Hz and heart-driven regular oscillations at about 1 Hz simultaneously. The results demonstrate the potential of the technique in monitoring both pulsating and steady components of the blood in inflammatory lesions of the skin.
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Special Section on Optical Diagnostic and Biophotonic Methods from Bench to Bedside
This PDF file contains the editorial “Special Section Guest Editorial: Optical Diagnostic and Biophotonic Methods from Bench to Bedside” for JBO Vol. 15 Issue 06
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Near-infrared spectroscopy is a novel imaging technique potentially sensitive to both brain hemodynamics (slow signal) and neuronal activity (fast optical signal, FOS). The big challenge of measuring FOS noninvasively lies in the presumably low signal-to-noise ratio. Thus, detectability of the FOS has been controversially discussed. We present reliable detection of FOS from 11 individuals concurrently with electroencephalogram (EEG) during a Go-NoGo task. Probes were placed bilaterally over prefrontal cortex. Independent component analysis (ICA) was used for artifact removal. Correlation coefficient in the best correlated FOS-EEG ICA pairs was highly significant (p < 10−8), and event-related optical signal (EROS) was found in all subjects. Several EROS components were similar to the event-related potential (ERP) components. The most robust "optical N200" at t = 225 ms coincided with the N200 ERP; both signals showed significant difference between targets and nontargets, and their timing correlated with subject's reaction time. Correlation between FOS and EEG even in single trials provides further evidence that at least some FOS components "reflect" electrical brain processes directly. The data provide evidence for the early involvement of prefrontal cortex in rapid object recognition. EROS is highly localized and can provide cost-effective imaging tools for cortical mapping of cognitive processes.
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TOPICS: Breast, Tissues, Tissue optics, Magnetic resonance imaging, Near infrared, Tumors, In vivo imaging, Image segmentation, Near infrared spectroscopy, Natural surfaces
We demonstrate quantitative functional imaging using image-guided near-infrared spectroscopy (IG-NIRS) implemented with the boundary element method (BEM) for reconstructing 3-D optical property estimates in breast tissue in vivo. A multimodality MRI-NIR system was used to collect measurements of light reflectance from breast tissue. The BEM was used to model light propagation in 3-D based only on surface discretization in order to reconstruct quantitative values of total hemoglobin (HbT), oxygen saturation, water, and scatter. The technique was validated in experimental measurements from heterogeneous breast-shaped phantoms with known values and applied to a total of seven subjects comprising six healthy individuals and one participant with cancer imaged at two time points during neoadjuvant chemotherapy. Using experimental measurements from a heterogeneous breast phantom, BEM for IG-NIRS produced accurate values for HbT in the inclusion with a <3% error. Healthy breast tissues showed higher HbT and water in fibroglandular tissue than in adipose tissue. In a subject with cancer, the tumor showed higher HbT compared to the background. HbT in the tumor was reduced by 9 µM during treatment. We conclude that 3-D MRI-NIRS with BEM provides quantitative and functional characterization of breast tissue in vivo through measurement of hemoglobin content. The method provides potentially complementary information to DCE-MRI for tumor characterization.
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We present polarization-sensitive optical coherence tomography (PS-OCT) for quantitative assessment of retinal pathologies in age-related macular degeneration (AMD). On the basis of the polarization scrambling characteristics of the retinal pigment epithelium, novel segmentation algorithms were developed that allow one to segment pathologic features such as drusen and atrophic zones in dry AMD as well as to determine their dimensions. Results from measurements in the eyes of AMD patients prove the ability of PS-OCT for quantitative imaging based on the retinal features polarizing properties. Repeatability measurements were performed in retinas diagnosed with drusen and geographic atrophy in order to evaluate the performance of the described methods. PS-OCT appears as a promising imaging modality for three-dimensional retinal imaging and ranging with additional contrast based on the structures' tissue-inherent polarization properties.
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Polarization-sensitive optical coherence tomography (PS-OCT) is known to be advantageous because of its additional tissue-specific contrast of the anterior eye. So far, this advantage has been shown only qualitatively. We evaluate the improved visibility afforded by 3-D PS corneal and anterior eye segment OCT (PS-CAS-OCT) in visualizing the trabecular meshwork (TM) based on statistical evidences. A total of 31 normal subjects participated in this study. The anterior eye segments of both the eyes of the subjects are scanned using a custom-made PS-CAS-OCT and the standard-scattering OCT (S-OCT) and polarization-sensitive phase-retardation OCT (P-OCT) images are obtained. Three graders grade the visibility of the TM using a four-leveled grading system. The intergrader agreement, intermodality differences, and interquadrant dependence of visibility are statistically examined. All three of three combinations of graders show substantial agreement in visibility with P-OCT (ρ = 0.74, 0.70, and 0.68, Spearman's correlation), while only one of three shows substantial agreement with S-OCT (ρ = 0.72). Significant dependence of the visibility on the modality (S-OCT versus P-OCT) and quadrants are found by the analysis of variance. A subsequent Wilcoxon signed-rank test reveals significantly improved visibility. PS-CAS-OCT may become a useful tool for screening angle-closure glaucoma.
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We describe the development, design, fabrication, and testing of an optical wire to assist in the surgical removal of small lesions during breast-conserving surgery. We modify a standard localization wire by adding a 200-µm optical fiber alongside it; the resulting optical wire fit through an 18 gauge needle for insertion in the breast. The optical wire is anchored in the lesion by a radiologist under ultrasonic and mammographic guidance. At surgery, the tip is illuminated with an eye-safe, red, HeNe laser, and the resulting glowball of light in the breast tissue surrounds the lesion. The surgeon readily visualizes the glowball in the operating room. This glowball provides sufficient feedback to the surgeon that it is used (1) to find the lesion and (2) as a guide during resection. Light-guided lumpectomy is a simple enhancement to traditional wire localization that could improve the current standard of care for surgical treatment of small, nonpalpable breast lesions.
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We present a novel technique for remote noncontact blood pulse pressure measurement. It is based on tracking both temporal and amplitude changes of reflected secondary speckle produced in human skin when illuminated by a laser beam. The implemented technique extracts the difference between the systolic and the diastolic blood pressure. Experimental results are presented showing good agreement when compared with conventional measurement methods.
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We present results of a clinical study on bedside perfusion monitoring of the human brain by optical bolus tracking. We measure the kinetics of the contrast agent indocyanine green using time-domain near-IR spectroscopy (tdNIRS) in 10 patients suffering from acute unilateral ischemic stroke. In all patients, a delay of the bolus over the affected when compared to the unaffected hemisphere is found (mean: 1.5 s, range: 0.2 s to 5.2 s). A portable time-domain near-IR reflectometer is optimized and approved for clinical studies. Data analysis based on statistical moments of time-of-flight distributions of diffusely reflected photons enables high sensitivity to intracerebral changes in bolus kinetics. Since the second centralized moment, variance, is preferentially sensitive to deep absorption changes, it provides a suitable representation of the cerebral signals relevant for perfusion monitoring in stroke. We show that variance-based bolus tracking is also less susceptible to motion artifacts, which often occur in severely affected patients. We present data that clearly manifest the applicability of the tdNIRS approach to assess cerebral perfusion in acute stroke patients at the bedside. This may be of high relevance to its introduction as a monitoring tool on stroke units.
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The concept of using diffuse reflectance spectroscopy to distinguish intraoperatively between pediatric brain tumors and normal brain parenchyma at the edge of resection cavities is evaluated using an in vivo human study. Diffuse reflectance spectra are acquired from normal and tumorous brain areas of 12 pediatric patients during their tumor resection procedures, using a spectroscopic system with a handheld optical probe. A total of 400 spectra are acquired at the rate of 33 Hz from a single investigated site, from which the mean spectrum and the standard deviation are calculated. The mean diffuse reflectance spectra collected are divided into the normal and the tumorous categories in accordance with their corresponding results of histological analysis. Statistical methods are used to identify those spectral features that effectively separated the two tissue categories, and to quantify the spectral variations induced by the motion of the handheld probe during a single spectral acquisition procedure. The results show that diffuse reflectance spectral intensities between 600 and 800 nm are effective in terms of differentiating normal cortex from brain tumors. Furthermore, probe movements induce large variations in spectral intensities (i.e., larger standard deviation) between 400 and 600 nm.
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By employing high-aspect-ratio parallel microchannels as an angular filter, quasiballistic photons sensitive to internal structures in a turbid medium can be captured. Scattered photons exiting the turbid medium typically exhibit trajectories with random angles compared to the initial trajectory and are mostly rejected by the filter. However, angular filter arrays cannot differentiate between quasiballistic photons (early arriving) and photons that happen to attain a scattered trajectory that is within the acceptance angle (late arriving). Therefore, we have two objectives: (1) to experimentally characterize the angular distribution and proportion of minimally deviated quasiballistic photons and multiply scattered photons in a turbid medium and (2) to combine time and angular gating principles so that early and late arriving photons can be distinguished. From the angular distribution data, the angular filter with angular acceptance about 0.4 deg yields the highest image contrast for transillumination images. The use of angular domain imaging(ADI) with time-gating enables visualization of submillimeter absorbing objects with approximately seven times higher image contrast compared to ADI in a turbid medium with a scattering level of six times the reduced mean free path.
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Coronary calcified plaque (CP) is both an important marker of atherosclerosis and major determinant of the success of coronary stenting. Intracoronary optical coherence tomography (OCT) with high spatial resolution can provide detailed volumetric characterization of CP. We present a semiautomatic method for segmentation and quantification of CP in OCT images. Following segmentation of the lumen, guide wire, and arterial wall, the CP was localized by edge detection and traced using a combined intensity and gradient-based level-set model. From the segmentation regions, quantification of the depth, area, angle fill fraction, and thickness of the CP was demonstrated. Validation by comparing the automatic results to expert manual segmentation of 106 in vivo images from eight patients showed an accuracy of 78±9%. For a variety of CP measurements, the bias was insignificant (except for depth measurement) and the agreement was adequate when the CP has a clear outer border and no guide-wire overlap. These results suggest that the proposed method can be used for automated CP analysis in OCT, thereby facilitating our understanding of coronary artery calcification in the process of atherosclerosis and helping guide complex interventional strategies in coronary arteries with superficial calcification.
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Anastomotic complication is a major morbidity associated with esophagectomy. Gastric ischemia after conduit creation contributes to anastomotic complications, but a reliable method to assess oxygenation in the gastric conduit is lacking. We hypothesize that fiber optic spectroscopy can reliably assess conduit oxygenation, and that intraoperative gastric ischemia will correlate with the development of anastomotic complications. A simple optical fiber probe spectrometer is designed for nondestructive laparoscopic measurement of blood content and hemoglobin oxygen saturation in the stomach tissue microvasculature during human esophagectomies. In 22 patients, the probe measured the light transport in stomach tissue between two fibers spaced 3-mm apart (500- to 650-nm wavelength range). The stomach tissue site of measurement becomes the site of a gastroesophageal anastamosis following excision of the cancerous esophagus and surgical ligation of two of the three gastric arteries that provide blood perfusion to the anastamosis. Measurements are made at each of five steps throughout the surgery. The resting baseline saturation is 0.51±0.15 and decreases to 0.35±0.20 with ligation. Seven patients develop anastomotic complications, and a decreased saturation at either of the last two steps (completion of conduit and completion of anastamosis) is predictive of complication with a sensitivity of 0.71 when the specificity equaled 0.71.
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In-vivo reflectance confocal microscopy (RCM) shows promise for the early detection of superficial spreading melanoma (SSM). RCM of SSM shows pagetoid melanocytes (PMs) in the epidermis and disarray at the dermal-epidermal junction (DEJ), which are automatically quantified with a computer algorithm that locates depth of the most superficial pigmented surface [DSPS(x,y)] containing PMs in the epidermis and pigmented basal cells near the DEJ. The algorithm uses 200 noninvasive confocal optical sections that image the superficial 200 μm of ten skin sites: five unequivocal SSMs and five nevi. The pattern recognition algorithm automatically identifies PMs in all five SSMs and finds none in the nevi. A large mean gradient ψ (roughness) between laterally adjacent points on DSPS(x,y) identifies DEJ disruption in SSM ψ = 11.7 ± 3.7 [−] for n = 5 SSMs versus a small ψ = 5.5 ± 1.0 [−] for n = 5 nevi (significance, p = 0.0035). Quantitative endpoint metrics for malignant characteristics make digital RCM data an attractive diagnostic asset for pathologists, augmenting studies thus far, which have relied largely on visual assessment.
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A new simple method for two-dimensional determination of optical density of macular pigment xanthophyll (ODx) in clinical routine is based on a single blue-reflection fundus image. Individual different vignetting is corrected by a shading function. For its construction, nodes are automatically found in structureless image regions. The influence of stray light in elderly crystalline lenses is compensated by a correction function that depends on age. The reproducibility of parameters in a one-wavelength reflection method determined for three subjects (47, 61, and 78 years old) was: maxODx = 6.3%, meanODx = 4.6%, volume = 6%, and area = 6% already before stray-light correction. ODx was comparable in pseudophakic and in an eye with a crystalline lens of the same 11 subjects after stray-light correction. Significant correlation in ODx was found between the one-wavelength reflection method and the two-wavelength autofluorescence method for pseudophakic and cataract eyes of 19 patients suffering from dry age-related macular degeneration (AMD) (R2 = 0.855). In pseudophakic eyes, maxODx was significantly lower for dry AMD (n = 45) (ODx = 0.491±0.102 ODU) than in eyes with healthy fundus (n = 22) (ODx = 0.615±0.103 ODU) (p = 0.000033). Also in eyes with crystalline lens, maxODx was lower in AMD (n = 125) (ODx = 0.610±0.093 ODU) than in healthy subjects (n = 45) (ODx = 0.674±0.098 ODU) (p = 0.00019). No dependence on age was found in the pseudophakic eyes both of healthy subjects and AMD patients.
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Foot ulcers affect millions of Americans annually. Conventional methods used to assess skin integrity, including inspection and palpation, may be valuable approaches, but they usually do not detect changes in skin integrity until an ulcer has already developed. We analyze the feasibility of thermal imaging as a technique to assess the integrity of the skin and its many layers. Thermal images are analyzed using an asymmetry analysis, combined with a genetic algorithm, to examine the infrared images for early detection of foot ulcers. Preliminary results show that the proposed technique can reliably and efficiently detect inflammation and hence effectively predict potential ulceration.
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Spatial frequency-domain imaging (SFDI) utilizes multiple-frequency structured illumination and model-based computation to generate two-dimensional maps of tissue absorption and scattering properties. SFDI absorption data are measured at multiple wavelengths and used to fit for the tissue concentration of intrinsic chromophores in each pixel. This is done with a priori knowledge of the basis spectra of common tissue chromophores, such as oxyhemoglobin (ctO2Hb), deoxyhemoglobin (ctHHb), water (ctH2O), and bulk lipid. The quality of in vivo SFDI fits for the hemoglobin parameters ctO2Hb and ctHHb is dependent on wavelength selection, fitting parameters, and acquisition rate. The latter is critical because SFDI acquisition time is up to six times longer than planar two-wavelength multispectral imaging due to projection of multiple-frequency spatial patterns. Thus, motion artifact during in vivo measurements compromises the quality of the reconstruction. Optimal wavelength selection is examined through matrix decomposition of basis spectra, simulation of data, and dynamic in vivo measurements of a human forearm during cuff occlusion. Fitting parameters that minimize cross-talk from additional tissue chromophores, such as water and lipid, are determined. On the basis of this work, a wavelength pair of 670 nm/850 nm is determined to be the optimal two-wavelength combination for in vivo hemodynamic tissue measurements provided that assumptions for water and lipid fractions are made in the fitting process. In our SFDI case study, wavelength optimization reduces acquisition time over 30-fold to 1.5s compared to 50s for a full 34-wavelength acquisition. The wavelength optimization enables dynamic imaging of arterial occlusions with improved spatial resolution due to reduction of motion artifacts.
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A photon-cell interactive Monte Carlo (pciMC) that tracks photon migration in both the extra- and intracellular spaces is developed without using macroscopic scattering phase functions and anisotropy factors, as required for the conventional Monte Carlos (MCs). The interaction of photons at the plasma-cell boundary of randomly oriented 3-D biconcave red blood cells (RBCs) is modeled using the geometric optics. The pciMC incorporates different photon velocities from the extra- to intracellular space, whereas the conventional MC treats RBCs as points in the space with a constant velocity. In comparison to the experiments, the pciMC yielded the mean errors in photon migration time of 9.8±6.8 and 11.2±8.5% for suspensions of small and large RBCs (RBCsmall, RBClarge) averaged over the optically diffusing region from 2000 to 4000 μm, while the conventional random walk Monte Carlo simulation gave statistically higher mean errors of 19.0±5.8 ( p < 0.047) and 21.7±19.1% (p < 0.055), respectively. The gradients of optical density in the diffusing region yielded statistically insignificant differences between the pciMC and experiments with the mean errors between them being 1.4 and 0.9% in RBCsmall and RBClarger, respectively. The pciMC based on the geometric optics can be used to accurately predict photon migration in the optically diffusing, turbid medium.
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Several studies have explored the potential of optoacoustic imaging for monitoring thermal therapies, yet the origin of the contrast in the images is not well understood. A technique is required to measure the changes in the optical and thermomechanical properties of tissues upon coagulation to better understand this contrast. An interferometric method is presented for measuring simultaneously the optical and thermomechanical properties of native and coagulated ex-vivo bovine tissue samples based on analysis of the surface displacement of irradiated samples. Surface displacements are measured after irradiation by short laser pulses at 750 nm. A 51% decrease in the optical attenuation depth is observed for coagulated liver samples compared to native samples. No significant differences in the Grüneisen coefficient are measured in the native and coagulated tissue samples. A mean value of 0.12 for the Grüneisen coefficient is measured for both native and coagulated liver tissues. The displacement profiles exhibit consistent differences between the two tissue types. To assess the changes in the sample mechanical properties, the experimental data also are compared to numerical solutions of the equation for thermoelastic deformation. The results demonstrate that differences in the tissue expansion dynamics arise from higher values of elastic modulus for coagulated liver samples compared to native ones.
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Laser pressure catapulting of adherent cells directly or cells grown on micropallets are two common methods of dislodgement. We describe a method where laser catapulting is performed as a flow is introduced orthogonally in a simple capillary chamber that is inexpensive and obviates flow-generating devices. The moving cells terminate near the contact line within the liquid medium, ensuring that they remain continuously hydrated and where the surface-tension forces hold them in place to permit a later collection process with a receptacle. By dislodging the cells close to the free edge of the liquid chamber, the amount of cell travel and, thus, contamination is minimized. The metrics of cell death and movement show that firing of the laser beam center a distance away from the cell to create a bubble that cavitates over time is more viable with the technique than directly on the cell.
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Microaneurysms (MAs) are the first manifestations of the diabetic retinopathy (DR) as well as an indicator for its progression. Their automatic detection plays a key role for both mass screening and monitoring and is therefore in the core of any system for computer-assisted diagnosis of DR. The algorithm basically comprises the following stages: candidate detection aiming at extracting the patterns possibly corresponding to MAs based on mathematical morphological black top hat, feature extraction to characterize these candidates, and classification based on support vector machine (SVM), to validate MAs. Feature vector and kernel function of SVM selection is very important to the algorithm. We use the receiver operating characteristic (ROC) curve to evaluate the distinguishing performance of different feature vectors and different kernel functions of SVM. The ROC analysis indicates the quadratic polynomial SVM with a combination of features as the input shows the best discriminating performance.
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Directional and nondirectional spectral reflection data from 0, 1, 2, 4, and 8 deg eccentricity, and the optic disk, were analyzed from 400 to 950 nm with an existing optical reflection model. The optical model, developed for the fovea, appeared to be also suitable for more eccentric locations. The optical densities of melanin and of the macular pigments zeaxanthin and lutein peaked in the fovea, in correspondence with literature data. The amplitude of the directional component, originating in the cone photoreceptors, had its maximum at 1 deg. The maximum of the directionality (peakedness) occurred at a slightly higher eccentricity.
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TOPICS: Monte Carlo methods, Picosecond phenomena, Tissue optics, Diffusion, Photon transport, Optical properties, Signal to noise ratio, Tomography, Data modeling, Pulsed laser operation
Time-resolved measurement of early arriving photons through diffusive media has been shown to effectively reduce the high degree of light scatter in biological tissue. However, the experimentally achievable reduction in photon scatter and the impact of time-gated detection on instrument noise performance is not well understood. We measure time-dependent photon density sensitivity functions (PDSFs) between a pulsed laser source and a photomultiplier tube operating in time-correlated single-photon-counting mode. Our data show that with our system, measurement of early arriving photons reduces the full width half maximum of PDSFs on average by about 40 to 60% versus quasicontinuous wave photons over a range of experimental conditions similar to those encountered in small animal tomography, corresponding to a 64 to 84% reduction in PDSF volume. Factoring in noise considerations, the optimal operating point of our instrument is determined to be about the 10% point on the rising edge of the transmitted intensity curve. Time-dependant Monte Carlo simulations and the time-resolved diffusion approximation are used to model photon propagation and are evaluated for agreement with experimental data.
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TOPICS: Sensors, Carbon monoxide, Signal processing, Wavelets, Infrared sensors, Temperature metrology, Signal detection, Light sources, Digital signal processing, Gas sensors
Most of the current respiratory devices for monitoring CO2 concentration use the side-stream structure. In this work, we engage to design a new double-end mainstream device for monitoring CO2 concentration of gas breathed out of the human body. The device can accurately monitor the cardiopulmonary status during anesthesia and mechanical ventilation in real time. Meanwhile, to decrease the negative influence of device noise and the low sample precision caused by temperature drift, wavelet packet denoising and temperature drift compensation are used. The new capnograph is proven by clinical trials to be helpful in improving the accuracy of capnography.
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A series of experiments are conducted in vivo using Yucatan mini-pigs (Sus scrofa domestica) to determine thermal damage thresholds to the skin from 1940-nm continuous-wave thulium fiber laser irradiation. Experiments employ exposure durations from 10 ms to 10 s and beam diameters of approximately 4.8 to 18 mm. Thermal imagery data provide a time-dependent surface temperature response from the laser. A damage endpoint of minimally visible effect is employed to determine threshold for damage at 1 and 24 h postexposure. Predicted thermal response and damage thresholds are compared with a numerical model of optical-thermal interaction. Results are compared with current exposure limits for laser safety. It is concluded that exposure limits should be based on data representative of large-beam exposures, where effects of radial diffusion are minimized for longer-duration damage thresholds.
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The study of short-pulse propagation through biological tissues is important due to the medical applications of short-pulse lasers. Techniques used for numerical study of short pulses through human tissues include the Monte Carlo (MC) method, the finite-element method, and the finite-difference time-domain (FDTD), but these are often time consuming. Recently, the boundary integral method (BIM) was applied to overcome this problem. The literature shows that the BIM is faster than the other mentioned methods. We first investigate the precision of results obtained by the BIM by comparison with those results obtained by the MC and FDTD methods. Then we use the BIM to investigate the short-pulse penetration into biological tissues. We also study the effects of optical properties of tissues such as scattering, the absorption coefficient, the anisotropic factor on the penetrating pulse. We also, consider the propagation of pulses emitted from extended sources with different temporal evolutions.
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We report results of a study on the use of Laguerre-Gaussian (LG) modes for optical trapping of spermatozoa. The results show that for a given trap beam power the first-order LG mode (LG01) leads to lower photodamage to the cells without compromising the trapping efficiency.
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Confocal Raman microspectroscopy is used to discriminate between different species of bacteria grown in biofilms. Tests are performed using two bacterial species, Streptococcus sanguinis and Streptococcus mutans, which are major components of oral plaque and of particular interest due to their association with healthy and cariogenic plaque, respectively. Dehydrated biofilms of these species are studied as a simplified model of dental plaque. A prediction model based on principal component analysis and logistic regression is calibrated using pure biofilms of each species and validated on pure biofilms grown months later, achieving 96% accuracy in prospective classification. When biofilms of the two species are partially mixed together, Raman-based identifications are achieved within ~2 µm of the boundaries between species with 97% accuracy. This combination of spatial resolution and predication accuracy should be suitable for forming images of species distributions within intact two-species biofilms.
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TOPICS: Calcium, Luminescence, Reconstruction algorithms, Action potentials, Neurons, Deconvolution, Signal to noise ratio, In vivo imaging, Convolution, Linear filtering
Identification of a small population of neuronal action potentials (APs) firing is considered essential to discover the operating principles of neuronal circuits. A promising method is to indirectly monitor the AP discharges in neurons from the recordings their intracellular calcium fluorescence transients. However, it is hard to reveal the nonlinear relationship between neuronal calcium fluorescence transients and the corresponding AP burst discharging. We propose a method to reconstruct the neuronal AP train from calcium fluorescence diversifications based on a multiscale filter and a convolution operation. Results of experimental data processing show that the false-positive rate and the event detection rate are about 10 and 90%, respectively. Meanwhile, the APs firing at a high frequency up to 40 Hz can also be successfully identified. From the results, it can be concluded that the method has strong power to reconstruct a neuronal AP train from a burst firing.
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We present a new method for quantitative visualization of premalignant oral epithelium called scattering attenuation microscopy (SAM). Using low-coherence interferometry, SAM projects measurements of epithelial optical attenuation onto an image of the tissue surface as a color map. The measured attenuation is dominated by optical scattering that provides a metric of the severity of oral epithelial dysplasia (OED). Scattering is sensitive to the changes in size and distribution of nuclear material that are characteristic of OED, a condition recognized by the occurrence of basal-cell-like features throughout the epithelial depth. SAM measures the axial intensity change of light backscattered from epithelial tissue. Scattering measurements are obtained from sequential axial scans of a 3-D tissue volume and displayed as a 2-D SAM image. A novel segmentation method is used to confine scattering measurement to epithelial tissue. This is applied to oral biopsy samples obtained from 19 patients. Our results show that imaging of tissue scattering can be used to discriminate between different dysplastic severities and furthermore presents a powerful tool for identifying the most representative tissue site for biopsy.
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We develop an adaptive optics (AO) multiphoton microscope by incorporating a deformable mirror and a Hartmann-Shack wavefront sensor. The AO module operating in closed-loop is used to correct for the aberrations of the illumination laser beam. This increases the efficiency of the nonlinear processes in reducing tissue photodamage, improves contrast, and enhances lateral resolution in images of nonstained ocular tissues. In particular, the use of AO in the multiphoton microscope provides a better visualization of ocular structures, which are relevant in ophthalmology. This instrument might be useful to explore the possible connections between changes in ocular structures and the associated pathologies.
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Localization of a single fluorescent molecule is required in a number of superresolution imaging techniques for visualizing biological structures at cellular and subcellular levels. The localization capability and limitation of low-light detectors are critical for such a purpose. We present an updated evaluation on the performance of three typical low-light detectors, including a popular electron-multiplying CCD (EMCCD), a newly developed scientific CMOS (sCMOS), and a representative cooled CCD, for superresolution imaging. We find that under some experimental accessible conditions, the sCMOS camera shows a competitive and even better performance than the EMCCD camera, which has long been considered the detector of choice in the field of superresolution imaging.
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We report the use of spectral Doppler optical coherence tomography imaging (SDOCTI) for quantitative evaluation of dynamic blood circulation before and after a localized ischemic stroke in a mouse model. Rose Bengal photodynamic therapy (PDT) is used as a noninvasive means for inducing localized ischemia in cortical microvasculature of the mouse. Fast, repeated Doppler optical coherence tomography scans across vessels of interest are performed to record flow dynamic information with high temporal resolution. Doppler-angle-independent flow indices are used to quantify vascular conditions before and after the induced ischemia by the photocoagulation of PDT. The higher (or lower) flow resistive indices are associated with higher (or lower) resistance states that are confirmed by laser speckle flow index maps (of laser speckle imaging). Our in vivo experiments shows that SDOCTI can provide complementary quantified flow information that is an alternative to blood volume measurement, and can be used as a means for cortical microvasculature imaging well suited for small animal studies.
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Tumor vascular patterns of advanced breast cancers are complex and heterogeneous. Two typical light absorption patterns of periphery enhancement and posterior shadowing have been observed when imaging these advanced cancers using optical tomography guided by ultrasound. We perform a series simulation and phantom experiments to systemically evaluate the effects of target parameters, target locations, and target optical properties on imaging periphery enhancement absorption distribution using reflection geometry. Large tumors are modeled as concentric semiellipsoidal targets of different outer shell and inner core optical properties. We show that larger targets of more than 3 to 4 cm diameter with outer shell thicknesses less than 1 cm can be resolved at a depth less than 3 cm. A clinical example is given to show the complex vasculature distributions seen from an advanced cancer.
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Hallmarks of high-fat Western diet intake, such as excessive lipid accumulation in skeletal muscle and liver as well as liver fibrosis, are investigated in tissues from mice using nonlinear microscopy, second harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS), supported by conventional analysis methods. Two aspects are presented; intake of standard chow versus Western diet, and a comparison between two high-fat Western diets of different polyunsaturated lipid content. CARS microscopy images of intramyocellular lipid droplets in muscle tissue show an increased amount for Western diet compared to standard diet samples. Even stronger diet impact is found for liver samples, where combined CARS and SHG microscopy visualize clear differences in lipid content and collagen fiber development, the latter indicating nonalcoholic fatty liver disease (NAFLD) and steatohepatitis induced at a relatively early stage for Western diet. Characteristic for NAFLD, the fibrous tissue-containing lipids accumulate in larger structures. This is also observed in CARS images of liver samples from two Western-type diets of different polyunsaturated lipid contents. In summary, nonlinear microscopy has strong potential (further promoted by technical advances toward clinical use) for detection and characterization of steatohepatitis already in its early stages.
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Diffuse optical tomographic image reconstruction uses advanced numerical models that are computationally costly to be implemented in the real time. The graphics processing units (GPUs) offer desktop massive parallelization that can accelerate these computations. An open-source GPU-accelerated linear algebra library package is used to compute the most intensive matrix-matrix calculations and matrix decompositions that are used in solving the system of linear equations. These open-source functions were integrated into the existing frequency-domain diffuse optical image reconstruction algorithms to evaluate the acceleration capability of the GPUs (NVIDIA Tesla C 1060) with increasing reconstruction problem sizes. These studies indicate that single precision computations are sufficient for diffuse optical tomographic image reconstruction. The acceleration per iteration can be up to 40, using GPUs compared to traditional CPUs in case of three-dimensional reconstruction, where the reconstruction problem is more underdetermined, making the GPUs more attractive in the clinical settings. The current limitation of these GPUs in the available onboard memory (4 GB) that restricts the reconstruction of a large set of optical parameters, more than 13,377.
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We demonstrate the use of tone-burst excitation and time-gated spectral analysis for photoacoustic Doppler mapping of flow in an unperturbed vessel phantom and in a vessel with a spatially varying lumen. The method, which mimics pulsed Doppler ultrasound, enables simultaneous measurement of axial position and flow as well as complete characterization of the Doppler spectrum over a wide range of mean velocities (3.5 to 200 mm/s). To generate the required optical excitation, a continuous cw laser source followed by an external electro-optic modulator is used. Stenoses at various levels are emulated in a C-flex tube with a flowing suspension of micrometer-scale carbon particles. Two-dimensional maps of spectral content versus axial position at different points along the vessel and for various levels of perturbations demonstrate the potential use of the method for characterization of flow irregularities.
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Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly--caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.
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Early detection is important for many solid cancers but the images provided by ultrasound, magnetic resonance imaging (MRI), and computed tomography applied alone or together, are often not sufficient for decisive early screening/diagnosis. We demonstrate that MRI augmented with fluorescence intensity (FI) substantially improves detection. Early stage murine pancreatic tumors that could not be identified by blinded, skilled observers using MRI alone, were easily identified with MRI along with FI images acquired with photomultiplier tube detection and offset laser scanning. Moreover, we show that fluorescence lifetime (FLT) imaging enables positive identification of the labeling fluorophore and discriminates it from surrounding tissue autofluorescence. Our data suggest combined-modality imaging with MRI, FI, and FLT can be used to screen and diagnose early tumors.
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Real-time grading of bladder urothelial carcinoma (UC) is clinically important, but the current standard for grading (histopathology) cannot provide this information. Based on optical coherence tomography (OCT)-measured optical attenuation (µt), the grade of bladder UC could potentially be assessed in real time. We evaluate ex vivo whether µt differs between different grades of UC and benign bladder tissue. Human bladder tissue specimens are examined ex vivo by 850-nm OCT using dynamic focusing. Three observers independently determine the µt from the OCT images, and three pathologists independently review the corresponding histology slides. For both methods, a consensus diagnosis is made. We include 76 OCT scans from 54 bladder samples obtained in 20 procedures on 18 patients. The median (interquartile range) µt of benign tissue is 5.75 mm−1 (4.77 to 6.14) versus 5.52 mm−1 (3.47 to 5.90), 4.85 mm−1 (4.25 to 6.50), and 5.62 mm−1 (5.01 to 6.29) for grade 1, 2, and 3 UC, respectively (p = 0.732). Interobserver agreement of histopathology is "substantial" [Kappa 0.62, 95% confidence interval (IC) 0.54 to 0.70] compared to "almost perfect" [interclass correlation coefficient (ICC) 0.87, 95% CI 0.80 to 0.92] for OCT. Quantitative OCT analysis (by µt) does not detect morphological UC changes. This may be due to factors typical for an ex-vivo experimental setting.
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To evaluate the severity of airway pathologies, quantitative dimensioning of airways is of utmost importance. Endoscopic vision gives a projective image and thus no true scaling information can be directly deduced from it. In this article, an approach based on an interferometric setup, a low-coherence laser source and a standard rigid endoscope is presented, and applied to hollow samples measurements. More generally, the use of the low-coherence interferometric setup detailed here could be extended to any other endoscopy-related field of interest, e.g., gastroscopy, arthroscopy and other medical or industrial applications where tri-dimensional topology is required. The setup design with a multiple fibers illumination system is presented. Demonstration of the method ability to operate on biological samples is assessed through measurements on ex vivo pig bronchi.
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Rapid Raman mapping has the potential to be used for automated histopathology diagnosis, providing an adjunct technique to histology diagnosis. The aim of this work is to evaluate the feasibility of automated and objective pathology classification of Raman maps using linear discriminant analysis. Raman maps of esophageal tissue sections are acquired. Principal component (PC)-fed linear discriminant analysis (LDA) is carried out using subsets of the Raman map data (6483 spectra). An overall (validated) training classification model performance of 97.7% (sensitivity 95.0 to 100% and specificity 98.6 to 100%) is obtained. The remainder of the map spectra (131,672 spectra) are projected onto the classification model resulting in Raman images, demonstrating good correlation with contiguous hematoxylin and eosin (HE) sections. Initial results suggest that LDA has the potential to automate pathology diagnosis of esophageal Raman images, but since the classification of test spectra is forced into existing training groups, further work is required to optimize the training model. A small pixel size is advantageous for developing the training datasets using mapping data, despite lengthy mapping times, due to additional morphological information gained, and could facilitate differentiation of further tissue groups, such as the basal cells/lamina propria, in the future, but larger pixels sizes (and faster mapping) may be more feasible for clinical application.
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Multispectral widefield optical imaging has the potential to improve early detection of oral cancer. The appropriate selection of illumination and collection conditions is required to maximize diagnostic ability. The goals of this study were to (i) evaluate image contrast between oral cancer/precancer and non-neoplastic mucosa for a variety of imaging modalities and illumination/collection conditions, and (ii) use classification algorithms to evaluate and compare the diagnostic utility of these modalities to discriminate cancers and precancers from normal tissue. Narrowband reflectance, autofluorescence, and polarized reflectance images were obtained from 61 patients and 11 normal volunteers. Image contrast was compared to identify modalities and conditions yielding greatest contrast. Image features were extracted and used to train and evaluate classification algorithms to discriminate tissue as non-neoplastic, dysplastic, or cancer; results were compared to histologic diagnosis. Autofluorescence imaging at 405-nm excitation provided the greatest image contrast, and the ratio of red-to-green fluorescence intensity computed from these images provided the best classification of dysplasia/cancer versus non-neoplastic tissue. A sensitivity of 100% and a specificity of 85% were achieved in the validation set. Multispectral widefield images can accurately distinguish neoplastic and non-neoplastic tissue; however, the ability to separate precancerous lesions from cancers with this technique was limited.
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We apply spectral hole burning (SHB)-aided detection in ultrasound-modulated optical tomography (UOT) to image optical heterogeneities in thick tissue-mimicking phantom samples and chicken breast tissue. The efficiency of SHB is improved by using a Tm3+:YAG crystal of higher doping concentration (2.0-atomic%) and a double-pass pumping configuration, in which the pump beam is transmitted through the crystal twice to burn a deeper spectral hole with the available optical intensity. With the improved SHB-UOT system, we image absorbing, scattering, and phase objects that are embedded in the middle plane of a 30-mm-thick phantom sample. The imaging resolution was 0.5 mm in the lateral direction, as defined by the focal width of the ultrasonic transducer, and 1.5 mm in the axial direction, as determined by the ultrasonic burst length. We also image two absorbing objects embedded in a 32-mm-thick chicken breast sample. The results suggest that the improved SHB-UOT system is one step closer to the practical optical imaging application in biological and clinical studies.
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We demonstrate that morphological features pertinent to a tissue's pathology may be ascertained from localized measures of broadband reflectance, with a mesoscopic resolution (100-μm lateral spot size) that permits scanning of an entire margin for residual disease. The technical aspects and optimization of a k-nearest neighbor classifier for automated diagnosis of pathologies are presented, and its efficacy is validated in 29 breast tissue specimens. When discriminating between benign and malignant pathologies, a sensitivity and specificity of 91 and 77% was achieved. Furthermore, detailed subtissue-type analysis was performed to consider how diverse pathologies influence scattering response and overall classification efficacy. The increased sensitivity of this technique may render it useful to guide the surgeon or pathologist where to sample pathology for microscopic assessment.
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A computer-aided interpretation approach is proposed to detect rheumatic arthritis (RA) in human finger joints using optical tomographic images. The image interpretation method employs a classification algorithm that makes use of a so-called self-organizing mapping scheme to classify fingers as either affected or unaffected by RA. Unlike in previous studies, this allows for combining multiple image features, such as minimum and maximum values of the absorption coefficient for identifying affected and not affected joints. Classification performances obtained by the proposed method were evaluated in terms of sensitivity, specificity, Youden index, and mutual information. Different methods (i.e., clinical diagnostics, ultrasound imaging, magnet resonance imaging, and inspection of optical tomographic images), were used to produce ground truth benchmarks to determine the performance of image interpretations. Using data from 100 finger joints, findings suggest that some parameter combinations lead to higher sensitivities, while others to higher specificities when compared to single parameter classifications employed in previous studies. Maximum performances are reached when combining the minimum/maximum ratio of the absorption coefficient and image variance. In this case, sensitivities and specificities over 0.9 can be achieved. These values are much higher than values obtained when only single parameter classifications were used, where sensitivities and specificities remained well below 0.8.
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The influence of the tumor microenvironment and hypoxia plays a significant role in determining cancer progression, treatment response, and treatment resistance. That the tumor microenvironment is highly heterogeneous with significant intratumor and intertumor variability presents a significant challenge in developing effective cancer therapies. Critical to understanding the role of the tumor microenvironment is the ability to dynamically quantify oxygen levels in the vasculature and tissue in order to elucidate the roles of oxygen supply and consumption, spatially and temporally. To this end, we describe the use of hyperspectral imaging to characterize hemoglobin absorption to quantify hemoglobin content and oxygen saturation, as well as dual emissive fluorescent/phosphorescent boron nanoparticles, which serve as ratiometric indicators of tissue oxygen tension. Applying these techniques to a window-chamber tumor model illustrates the role of fluctuations in hemoglobin saturation in driving changes in tissue oxygenation, the two being significantly correlated (r = 0.77). Finally, a green-fluorescence-protein reporter for hypoxia inducible factor-1 (HIF-1) provides an endpoint for hypoxic stress in the tumor, which is used to demonstrate a significant association between tumor hypoxia dynamics and HIF-1 activity in an in vivo demonstration of the technique.
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Hemodynamics is thought to play a major role in heart development, yet tools to quantitatively assess hemodynamics in the embryo are sorely lacking. The especially challenging analysis of hemodynamics in the early embryo requires new technology. Small changes in blood flow could indicate when anomalies are initiated even before structural changes can be detected. Furthermore, small changes in the early embryo that affect blood flow could lead to profound abnormalities at later stages. We present a demonstration of 4-D Doppler optical coherence tomography (OCT) imaging of structure and flow, and present several new hemodynamic measurements on embryonic avian hearts at early stages prior to the formation of the four chambers. Using 4-D data, pulsed Doppler measurements could accurately be attained in the inflow and outflow of the heart tube. Also, by employing an en-face slice from the 4-D Doppler image set, measurements of stroke volume and cardiac output are obtained without the need to determine absolute velocity. Finally, an image plane orthogonal to the blood flow is used to determine shear stress by calculating the velocity gradient normal to the endocardium. Hemodynamic measurements will be crucial to identifying genetic and environmental factors that lead to congenital heart defects.
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Extremely low birth weight (ELBW) infants frequently require endotracheal intubation for assisted ventilation or as a route for administration of drugs or exogenous surfactant. In adults and less premature infants, the risks of this intubation can be greatly reduced using video laryngoscopy, but current products are too large and incorrectly shaped to visualize an ELBW infant's airway anatomy. We design and prototype a video laryngoscope using a miniature camera set in a curved acrylic blade with a 3×6-mm cross section at the tip. The blade provides a mechanical structure for stabilizing the tongue and acts as a light guide for an LED light source, located remotely to avoid excessive local heating at the tip. The prototype is tested on an infant manikin and found to provide sufficient image quality and mechanical properties to facilitate intubation. Finally, we show a design for a neonate laryngoscope incorporating a wafer-level microcamera that further reduces the tip cross section and offers the potential for low cost manufacture.
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There are vibrant developments of optical imaging systems and contrast-enhancing methods that are geared to enhancing surgical vision and the outcome of surgical procedures. Such optical technologies designed for intraoperative use can offer high integration in the operating room compared to conventional radiological modalities adapted to intraoperative applications. Simple fluorescence epi-illumination imaging, in particular, appears attractive but may lead to inaccurate observations due to the complex nature of photon-tissue interaction. Of importance therefore are emerging methods that account for the background optical property variation in tissues and can offer accurate, quantitative imaging that eliminates the appearance of false negatives or positives. In parallel, other nonfluorescent optical imaging methods are summarized and overall progress in surgical optical imaging applications is outlined. Key future directions that have the potential to shift the paradigm of surgical health care are also discussed.
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An imaging system for brain oxygenation based on a time-gated, intensified charge-coupled device camera was developed. It allows one to image diffusely reflected light from an investigated medium at defined time windows delayed with respect to the laser pulse. Applying a fast optomechanical switch to deliver the light at a wavelength of 780 nm to nine source fibers allowed one to acquire images in times as short as 4 s. Thus, the system can be applied in in vivo studies. The system was validated in phantom experiments, in which absorbing inclusions were localized at different depths and different lateral positions. Then, the decrease in absorption of the brain tissue related to increase in oxygenation was visualized in the motor cortex area during finger tapping by a healthy volunteer.
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Different colors of visible light penetrate to varying depths in tissue due to the wavelength dependence of tissue optical absorption and elastic scattering. We exploit this to map the contour of the closest surface of a buried fluorescent object. This uses a novel algorithm based on the diffusion theory description of light propagation in tissue at each excitation wavelength to derive metrics that define the depth of the top surface of the object. The algorithm was validated using a tissue-simulating phantom. It was then demonstrated in vivo by subsurface brain tumor topography in a rodent model, using the fluorescence signal from protoporphyrin IX that is preferentially synthesized within malignant cells following systemic application of aminolevulinic acid. Comparisons to histomorphometry in the brain post mortem show the spatial accuracy of the technique. This method has potential for fluorescence image-guided tumor surgery, as well as other biomedical and nonbiological applications in subsurface sensing.
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We report the numerical analysis of gradient index (GRIN) lens-based optical coherence tomography imaging probes to derive optimal design parameters. Long and short working distance probes with a small focal spot are considered. In each model, the working distance and beam waist are characterized and compared for different values of length and refractive index of the probe components. We also explore the influence of the outer tubing and refractive index of the sample media. Numerical results show that the adjustment of the maximum beam diameter and focusing angle at the end of the GRIN lens surface is very important for determining the optical performance parameters of the probe.
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Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APCMin mouse model and human cytology specimens of colorectal cancer. We show the ability of insitu nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as "indeterminate or normal" by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.
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We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.
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Monitoring cerebral blood flow (CBF) during neurosurgery can provide important physiological information for a variety of surgical procedures. CBF measurements are important for assessing whether blood flow has returned to presurgical baseline levels and for assessing postsurgical tissue viability. Existing techniques for intraoperative monitoring of CBF based on magnetic resonance imaging are expensive and often impractical, while techniques such as indocyanine green angiography cannot produce quantitative measures of blood flow. Laser speckle contrast imaging (LSCI) is an optical technique that has been widely used to quantitatively image relative CBF in animal models in vivo. In a pilot clinical study, we adapted an existing neurosurgical operating microscope to obtain LSCI images in humans in real time during neurosurgery under baseline conditions and after bipolar cautery. Simultaneously recorded ECG waveforms from the patient were used to develop a filter that helped reduce measurement variabilities due to motion artifacts. Results from this study demonstrate the feasibility of using LSCI to obtain blood flow images during neurosurgeries and its capability to produce full field CBF image maps with excellent spatial resolution in real-time with minimal disruption to the surgical procedure.
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Vision is initiated in photoreceptor cells of the retina by a set of biochemical events called phototransduction. These events occur via coordinated dynamic processes that include changes in secondary messenger concentrations, conformational changes and post-translational modifications of signaling proteins, and protein-protein interactions between signaling partners. A complete description of the orchestration of these dynamic processes is still unavailable. Described in this work is the first step in the development of tools combining fluorescent protein technology, Förster resonance energy transfer (FRET), and transgenic animals that have the potential to reveal important molecular insights about the dynamic processes occurring in photoreceptor cells. We characterize the fluorescent proteins SCFP3A and SYFP2 for use as a donor-acceptor pair in FRET assays, which will facilitate the visualization of dynamic processes in living cells. We also demonstrate the targeted expression of these fluorescent proteins to the rod photoreceptor cells of Xenopus laevis, and describe a general method for detecting FRET in these cells. The general approaches described here can address numerous types of questions related to phototransduction and photoreceptor biology by providing a platform to visualize dynamic processes in molecular detail within a native context.
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TOPICS: Near infrared, Tissues, Tumor growth modeling, Near infrared spectroscopy, Spectroscopy, Fuzzy logic, Diagnostics, Data modeling, Statistical modeling, Fuzzy systems
NIR spectra of 77 endometrium sections (malignant, hyperplasia, and normal samples) are collected. Partial least squares discriminant analysis (PLS-DA) and fuzzy rule-building expert systems (FuRES) are used for classification based on the NIR spectral data. The classification ability of two classifiers is evaluated by using ten bootstraps and five Latin partitions. The results indicate that the classification ability of FuRES is better than that of PLS-DA. The sensitivity, specificity, and accuracy obtained from FuRES for malignant endometrium diagnosis are 90.0±0.7, 95.0±0.8, and 93.1±0.8%, respectively. The results demonstrate that NIR spectroscopy combined with the FuRES technique is promising for the classification of endometrial specimens and for practical diagnostic applications.
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The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months old), and adult (6 to 10 years old) horses. Spatial collagen content and collagen cross-link ratio are quantified from the spectra. Additionally, normalized second derivative spectra of samples are clustered using the k-means clustering algorithm. In quantitative analysis, collagen content in the subchondral bone increases rapidly between the newborn and immature horses. The collagen cross-link ratio increases significantly with age. In qualitative analysis, clustering is able to separate newborn and adult samples into two different groups. The immature samples display some nonhomogeneity. In conclusion, this is the first study showing that FTIR spectral imaging combined with clustering techniques can detect quantitative and qualitative changes in the collagen matrix of subchondral bone during growth and maturation.
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Recently many software-based approaches have been suggested for improving the range and accuracy of Hartmann-Shack aberrometry. We compare the performance of four representative algorithms, with a focus on aberrometry for the human eye. Algorithms vary in complexity from the simplistic traditional approach to iterative spline extrapolation based on prior spot measurements. Range is assessed for a variety of aberration types in isolation using computer modeling, and also for complex wavefront shapes using a real adaptive optics system. The effects of common sources of error for ocular wavefront sensing are explored. The results show that the simplest possible iterative algorithm produces comparable range and robustness compared to the more complicated algorithms, while keeping processing time minimal to afford real-time analysis.
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A combination tapered fiber-optic biosensor (CTFOB) dip probe for rapid and cost-effective quantification of proteins in serum samples has been developed. This device relies on diode laser excitation and a charged-coupled device spectrometer and functions on a technique of sandwich immunoassay. As a proof of principle, this technique was applied in a quantitative estimation of interleukin IL-6. The probes detected IL-6 at picomolar levels in serum samples obtained from a patient with lupus, an autoimmune disease, and a patient with lymphoma. The estimated concentration of IL-6 in the lupus sample was 5.9 ± 0.6 pM, and in the lymphoma sample, it was below the detection limit. These concentrations were verified by a procedure involving bead-based xMAP technology. A similar trend in the concentrations was observed. The specificity of the CTFOB dip probes was assessed by analysis with receiver operating characteristics. This analysis suggests that the dip probes can detect 5-pM or higher concentration of IL-6 in these samples with specificities of 100%. The results provide information for guiding further studies in the utilization of these probes to quantify other analytes in body fluids with high specificity and sensitivity.
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We present a method for tissue fluorescence quantification in situ using a handheld fiber optic probe that measures both the fluorescence and diffuse reflectance spectra. A simplified method to decouple the fluorescence spectrum from distorting effects of the tissue optical absorption and scattering is developed, with the objective of accurately quantifying the fluorescence in absolute units. The primary motivation is measurement of 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) concentration in tissue during fluorescence-guided resection of malignant brain tumors. This technique is validated in phantoms and ex vivo mouse tissues, and tested in vivo in a rabbit brain tumor model using ALA-PpIX fluorescence contrast.
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The present work deals with the evaluation of a high-performance liquid chromatography laser-induced fluorescence (HPLC-LIF) technique developed in our laboratory for early detection of oral cancer from protein profiles of body fluids. The results show that protein profiles of serum samples from a given class of samples, say, normal, premalignant, or malignant, are statistically very close to each other, while profiles of members of any class are significantly different from other classes. The performance of the technique is evaluated by the use of sensitivity and specificity pairs, receiver operating characteristic (ROC) analysis, and Youden's Index. The technique uses protein profile differences in serum samples, registered by the HPLC-LIF technique. The study is carried out using serum samples from volunteers diagnosed as normal or premalignant clinically, and as malignant by histopathology. The specificities and sensitivities of the HPLC-LIF method at an ideal threshold (M-distance = 2) for normal, malignant, and premalignant classes are 100, 69.5, and 61.5%, and 86.5, 87.5, and 87.5% respectively.
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We report on development of dual-trap Raman tweezers for monitoring cellular dynamics and heterogeneity of interacting living cells suspended in a liquid medium. Dual-beam optical tweezers were combined with Raman spectroscopy, which allows capturing two cells that are in direct contact or closely separated by a few micrometers and simultaneously acquiring their Raman spectra with an imaging CCD spectrograph. As a demonstration, we recorded time-lapse Raman spectra of budding yeast cells held in dual traps for over 40 min to monitor the dynamic growth in a nutrient medium. We also monitored two germinating Bacillus spores after the initiation with L-alanine and observed their heterogeneity in the release of CaDPA under identical microenvironment.
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We present a new variant of a noncontact, oblique incidence spatially resolved reflectance setup. The continuously variable source detector separation enables adaptation to high and low albedo samples. Absorption (μa) and reduced scattering coefficients (μ′s) are determined in the wavelength range of 400-1000 nm using a lookup table, calculated by a Monte Carlo simulation of the light transport. The method is characterized by an silicone phantom study covering a wide parameter range 0.01 mm−;1 ≤ μa ≤ 2.5 mm−1 and 0.2 mm−1 ≤ μ′s ≤ 10 mm−1, which includes the optical parameters of tissue in the visible and near infrared. The influence of the incident angle and the detection aperture on the simulated remission was examined. Using perpendicular incidence and 90-deg detection aperture in the Monte Carlo simulation in contrast to the experimental situation with 30-deg incidence and 4.6-deg detection aperture is shown to be valid for the parameter range μ′s > 1 mm−;1 and μa < 1.2 mm−;1. A Mie calculation is presented, showing that a decreasing reduced scattering coefficient for increasing absorption can be the consequence of real physics instead of cross talk.
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Raman spectroscopy is used to study the effects of heating on specific molecular bonds present in albumen-based coagulation phantoms and ex-vivo tissues. Thermal coagulation is induced by submerging albumen-based phantoms in a 75°C water bath to achieve target temperatures of 45, 55, 65, and 75°C. Laser photocoagulation is performed on ex-vivo bovine muscle samples, yielding induced temperatures between 46 and 90°C, as reported by implanted microthermocouples. All phantoms and tissue samples are cooled to room temperature, and Raman spectra are acquired at the microthermocouple locations. Shifts in major Raman bands are observed with laser heating in bovine muscle, specifically from the amide-1 -helix group (~1655 cm−1), the CH2/CH3 group (~1446 cm−1), the C-H stretch group (~1312 cm−1), and the CN stretch group (~1121cm−1). Raman bands at 1334 cm−1 (tryptophan), 1317 cm−1C-H, and 1655 cm−1 (amide-1 -helix) also show a decrease in intensity following heating. The results suggest that Raman band locations and relative intensities are affected by thermal denaturation of proteins, and hence, may be a useful tool for monitoring the onset and progression of coagulation during thermal therapies.
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Despite the emergence of nonablative fractional resurfacing (NFR) as a new therapeutic modality for skin photoaging, little is known about the molecular events that underlie the heat shock response to different treatment parameters. Human subjects are treated with a scanned 1550-nm fractional laser at pulse energies spanning 6 to 40 mJ and a 140-µm spot size. The heat shock response is assessed immunohistochemically immediately through 7 days posttreatment. At the immediately posttreatment time point, we observe subepidermal clefting in most sections. The basal epidermis and dermal zones of sparing are both found to express HSP47, but not HSP72. By day 1, expression of HSP72 is detected throughout the epidermis, while that of HSP47 remains restricted to the basal layer. Both proteins are detected surrounding the dermal portion of the microscopic treatment zone (MTZ). This pattern of expression persists through day 7 post-NFR, although neither protein is found within the MTZ. Immediately posttreatment, the mean collagen denaturation zone width is 50 µm at 6 mJ, increasing to 202 µm at 40 mJ. The zone of cell death exceeds the denaturation zone by 19 to 55% over this pulse energy range. The two zones converge by day 7 posttreatment.
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We present a high-speed photographic analysis of the interaction of cavitation bubbles generated in two spatially separated regions by femtosecond laser-induced optical breakdown in water. Depending on the relative energies of the femtosecond laser pulses and their spatial separation, different kinds of interactions, such as a flattening and deformation of the bubbles, asymmetric water flows, and jet formation were observed. The results presented have a strong impact on understanding and optimizing the cutting effect of modern femtosecond lasers with high repetition rates (>1 MHz).
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