A clinical trial comprising 334 pigmented and vascular lesions has been performed in three Riga clinics by means of multispectral imaging analysis. The imaging system Nuance 2.4 (CRi) and self-developed software for mapping of the main skin chromophores were used. Specific features were observed and analyzed for malignant skin melanomas: notably higher absorbance (especially as the difference of optical density relative to the healthy skin), uneven chromophore distribution over the lesion area, and the possibility to select the "melanoma areas" in the correlation graphs of chromophores. The obtained results indicate clinical potential of this technology for noncontact selection of melanoma from other pigmented and vascular skin lesions.

Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.

We demonstrate speckle reduction based on angular compounding using parallel-detection spectral-domain optical coherence tomography (OCT). An ultrahigh-speed two-dimensional complementary metal-oxide semiconductor camera acquired angular and spectral interference fringes (128×1024 pixels) simultaneously at 15,000 frames/s for a single lateral point. A signal-to-noise ratio improvement of 8 dB was achieved for imaging human skin in vivo by averaging 121 angle-resolved OCT images.

We have developed a miniature integrated optical coherence tomography (OCT) ultrasound (US) probing system for intravascular imaging applications. In the OCT probe, the light coming out of a single mode fiber is focused by a gradient-index lens and then reflected by a right-angle prism from the side of the probe into the sample. It was combined with a 35 MHz PMN-PT side-viewing ultrasound transducer to obtain the ultrasound image as well. The OCT and ultrasound probes were integrated as a single probe to obtain OCT and ultrasound images simultaneously. The integrated probe has an outer diameter of 0.69 mm which, to our knowledge, is the smallest integrated OCT-US probe reported. Fast data acquisition and processing was implemented for real-time imaging. In vitro OCT and US images of human coronary artery with pathology, as well as in vivo images of normal rabbit abdominal aorta, were obtained using the integrated OCT-US probe to demonstrate its capability.

We propose a useful method to boost the imaging speed for spectral domain optical coherence tomography (SDOCT) by multiplying a number of high-speed spectrometers used in the system with selective precise control of data-recording and data-reading phases for spectral cameras employed in each spectrometer. To demonstrate the proposed method, we use two spectrometers built in a 1310 nm-band SDOCT system, each equipped with a high-speed InGaAs line-scan camera capable of 92-kHz line-scan rate, to achieve an unprecedented imaging speed at 184,000 lines/s. We validate the multiplied imaging speed by measuring Doppler-induced phase shift in the spectrograms using a flow phantom.

We investigated the use of a near-infrared (NIR) fluorescent dye, Rhodamine 800 (Rhod800, λ_exc = 693 nm, λ_em > 720 nm) as a flow-dependent molecular tracer for NIR spectroscopy and high-resolution cardiac imaging. Rhod800 accumulates in isolated mitochondria in proportion to the mitochondrial membrane potential (Δψ). However, in the intact myocardium, Rhod800 binding is Δψ-independent. Rat hearts were perfused in a Langendorff mode with Krebs-Henseleit buffer containing 45-nM Rhod800 at normal (100%), increased (150%), or reduced (50%) baseline coronary flow (CF) per gram, for 30 to 60 min. In a different group of hearts, the left anterior descending artery (LAD) was occluded prior to Rhod800 infusion to create a flow deficit area. Rhod800 deposition was analyzed by: 1. absorbance spectroscopy kinetics in the Rhod800-perfused hearts, 2. Rhod800 absorbance and fluorescence imaging in the short-axis heart slices, and 3. dynamic epicardial/subepicardial fluorescence imaging of Rhod800 in KCl-arrested hearts, with a spatial resolution of ~200µm. Rhod800 deposition was proportional to the perfusate volume (CF and perfusion time) and there was no Rhod800 loss during the washout period. In the LAD-ligated hearts, Rhod800 fluorescence was missing from the no-flow, LAD-dependent endocardial and epicardial/subepicardial area. We concluded that Rhod800 can be used as a deposition flow tracer for dynamic cardiac imaging.

The design and testing of a pneumatic optical positioning interface produced with the goal of improving fiber positioning in magnetic resonance (MR)-guided diffuse spectral imaging of breast cancer is presented. The system was created for vertical positioning of optical fibers inside the MR bore during a patient exam to target suspicious lesions with MR scans for reference and collect multiple planes of optical data. The interface includes new fiber plates for mechanical and optical coupling to the breast, and was tested in phantoms and human imaging. Reconstructions with data taken in the new interface show acceptable linearity over different absorber concentrations (residual norm = 0.067), and exhibit good contrast recovery at different imaging planes, which is consistent with previous work. An example of human breast imaging through the new interface is shown and a discussion of how it compares to other patient interfaces for breast imaging is presented. Design goals of increasing the available degrees of freedom for fiber positioning while maintaining good patient-fiber contact and comfort were accomplished. This interface allows improved volumetric imaging with interactive and accurate slice selection to quantify targeted suspicious lesions.

Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1′-bis-(4-sulfobutyl) indotricarbocyanine-5,5′-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-µm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm^-1 at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation.

In-flight insect wing motion behavior depends on a wide variety of conditions. They have a complex structural system and what seems to be a rather complicated motion. Researchers in many fields have endeavoured to study and reproduce these wing movements with the aim to apply the gained knowledge in their fields and for the benefit of avionic technological improvements and insect migration studies, among many other themes. The study of in-flight insect wing motion and its measurement is a relevant issue to understand and reproduce its functionality. Being capable of measuring the wing flapping using optical noninvasive methods adds scientific and technological value to the fundamental research in the area. Four different types of butterflies found widely in Mexico's forests are used to compare their wing flapping mechanisms. An out-of-plane digital holographic interferometry system is used to detect and measure its wingmicro deformations. Displacement changes from in vivo flapping wings are registered with a CMOS high speed camera yielding full field of view images depicting these insects' wing motion. The results have a resolution in the scale of hundreds of nanometers over the entire wing surface.

We investigate the processes of laser radiation transformation by biological crystal networks using the singular optics techniques. The obtained results show a distinct correlation between the points of "characteristic" values of coordinate distributions of Mueller matrix (M_ik = 0, ±1) elements and polarization singularities (L- and C-points) in laser images of biological crystal networks with the following possibility of Mueller-matrix selection of polarization singularity. The technique of Mueller-matrix singular diagnostics of pathological changes in a woman's reproductive sphere tissue (myometrium) is proposed.

We report a novel in vivo spectral imaging approach to cancer detection and chemotherapy assessment. We describe and characterize a ratiometric spectral imaging and analysis method and evaluate its performance for tumor detection and delineation by quantitatively monitoring the specific accumulation of targeted gallium corrole (HerGa) into HER2-positive (HER2 +) breast tumors. HerGa temporal accumulation in nude mice bearing HER2 + breast tumors was monitored comparatively by a. this new ratiometric imaging and analysis method; b. established (reflectance and fluorescence) spectral imaging; c. more commonly used fluorescence intensity imaging. We also tested the feasibility of HerGa imaging in vivo using the ratiometric spectral imaging method for tumor detection and delineation. Our results show that the new method not only provides better quantitative information than typical spectral imaging, but also better specificity than standard fluorescence intensity imaging, thus allowing enhanced in vivo outlining of tumors and dynamic, quantitative monitoring of targeted chemotherapy agent accumulation into them.

The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images.

We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.

The accurate and rapid assessment of tumor margins during breast cancer resection using optical coherence tomography (OCT) has the potential to reduce patient risk. However, it is difficult to subjectively distinguish cancer from normal fibroglandular stromal tissues in OCT images, and an objective measure is needed. In this initial study, we investigate the potential of a one-dimensional fractal box-counting method for cancer classification in OCT. We computed the fractal dimension, a measure of the self-similarity of an object, along the depth axis of 44 ultrahigh-resolution OCT images of human breast tissues obtained from 4 cancer patients. Correlative histology was employed to identify distinct regions of adipose, stroma, and cancer in the OCT images. We report that the fractal dimension of stroma is significantly higher than that of cancer (P < 10^-5, t-test). Furthermore, by adjusting the cutoff values of fractal dimension between cancer, stroma, and adipose tissues, sensitivities and specificities of either 82.4% and 88.9%, or 88.2% and 81.5%, are obtained, respectively, for cancer classification. The use of fractal analysis with OCT could potentially provide automated identification of tumor margins during breast-sparing surgery.

This paper reports the feasibility of optical coherence tomography (OCT) technology for inspection of bonding quality of microfluidic devices in manufacturing environments. A compact optical-fiber-based OCT is developed and its measurement performance is characterized. A series of microfluidic devices respectively bonded by adhesive tape, thermal method, and oxygen plasma, are inspected. The defects of geometry deformation and sealing completeness are emphasized during measurements. Based on the inspection results, some discoveries related to the production of microfluidic devices are discussed.

A simple method to fabricate Eu3⁺ doped silica nanoshells particles with 100 and 200 nm diameters is reported. Amino polystyrene beads were used as templates, and an 8 to 10 nm thick silica gel coating was formed by the sol-gel reaction. After removing the template by calcination, porous dehydrated silica gel nanoshells of uniform size were obtained. The Eu3⁺ doped silica nanoshells exhibited a red emission at 615 nm on UV excitation. The porous structure of the silica shell wall was characterized by transmission electron microscopy measurements, while particle size and zeta potentials of the particles suspended in aqueous solution were characterized by dynamic light scattering. Two-photon microscopy was used to image the nanoshells after assimilation by HeLa cancer cells.

The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 − 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼200 μm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.

The nanophosphors, or other similar materials, emit near-infrared (NIR) light upon x-ray excitation. They were designed as optical probes for in vivo visualization and analysis of molecular and cellular targets, pathways, and responses. Based on the previous work on x-ray fluorescence computed tomography (XFCT) and x-ray luminescence computed tomography (XLCT), here we propose a spectrally-resolving and scattering-compensated x-ray luminescence/fluorescence computed tomography (SXLCT or SXFCT) approach to quantify a spatial distribution of nanophosphors (other similar materials or chemical elements) within a biological object. In this paper, the x-ray scattering is taken into account in the reconstruction algorithm. The NIR scattering is described in the diffusion approximation model. Then, x-ray excitations are applied with different spectra, and NIR signals are measured in a spectrally resolving fashion. Finally, a linear relationship is established between the nanophosphor distribution and measured NIR data using the finite element method and inverted using the compressive sensing technique. The numerical simulation results demonstrate the feasibility and merits of the proposed approach.

Autofluorescence endoscopy is a promising functional imaging technique to improve screening of pre-cancerous or early cancer lesions in the gastrointestinal (GI) tract. Tissue autofluorescence signal is weak compared to white light reflectance imaging. Conventional forward-viewing endoscopes are inefficient in the collection of light from objects of interest along on the GI luminal wall. A key component of a complete autofluorescence endoscope is the light collection module. In this paper, we report the design, optimization, prototype development, and testing of an endoscope objective that is capable of acquiring simultaneous forward and radial views. The radial-view optical design was optimized for a balance between image quality and light collection. Modulation transfer function (MTF), entrance pupil radius, manufacturability, and field-of-view were parameters used in the lens optimization. In comparison with the typical forward-viewing endoscopes, our nonsequential ray trace simulations suggest the proposed radial-view design is more practical in the light collection. To validate the proposed simulation methods, a 3:1 scaled-up prototype was fabricated. Contrast measurements were taken with the prototype, and then compared with the simulated MTF.

Here we present an ultrahigh-speed Fourier-domain optical coherence tomography (OCT) that records the OCT spectrum in streak mode with a high-speed area scan camera, which allows higher OCT imaging speed than can be achieved with a line-scan camera. Unlike parallel OCT techniques that also use area scan cameras, the conventional single-mode fiber-based point-scanning mechanism is retained to provide a confocal gate that rejects multiply scattered photons from the sample. When using a 1000 Hz resonant scanner as the streak scanner, 1,016,000 A-scans have been obtained in 1 s. This method's effectiveness has been demonstrated by recording in vivo OCT-image sequences of embryonic chick hearts at 1000 frames/s. In addition, 2-megahertz OCT data have been obtained with another high speed camera.

Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

Optical coherence microscopy (OCM) combines coherence gating, high numerical aperture optics, and a fiber-core pinhole to provide high axial and lateral resolution with relatively large depth of imaging. We present a handheld rigid OCM endoscope designed for small animal surgical imaging, with a 6-mm diam tip, 1-mm scan width, and 1-mm imaging depth. X-Y scanning is performed distally with mirrors mounted to micro galvonometer scanners incorporated into the endoscope handle. The endoscope optical design consists of scanning doublets, an afocal Hopkins relay lens system, a 0.4 numerical aperture water immersion objective, and a cover glass. This endoscope can resolve laterally a 1.4-μm line pair feature and has an axial resolution (full width half maximum) of 5.4 μm. Images taken with this endoscope of fresh ex-vivo mouse ovaries show structural features, such as corpus luteum, primary follicles, growing follicles, and fallopian tubes. This rigid handheld OCM endoscope can be useful for a variety of minimally invasive and surgical imaging applications.

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.

For the first time, we have implemented photoacoustic tomography (PAT) to image the water content of an edema in vivo. We produced and imaged a cold-induced cerebral edema transcranially, then obtained blood vessel and water accumulation images at 610 and 975 nm, respectively. We tracked the changes at 12, 24, and 36 h after the cold injury. The blood volume decreased after the cold injury, and the maximum area of edema was observed 24 h after the cold injury. We validated PAT of the water content of the edema through magnetic Resonance Imaging and the water spectrum from the spectrophotometric measurement.

Photoacoustic spectra of normal, benign, and malignant ovarian tissues are recorded using 325-nm pulsed laser excitation in vitro. A total of 102 (34 normal, 38 benign, and 30 malignant) spectra are obtained from 22 samples belonging to normal, benign, and malignant subjects. Applying multi-algorithm approach, comprised of methods such as, principal component analysis (PCA) based k-nearest neighbor (k-NN) analysis, artificial neural network (ANN) analysis, and support vector machine (SVM) analysis, classification of the data has been carried out. For PCA, first the calibration set is formed by pooling 45 spectra, 15 belonging to each of pathologically certified normal, benign, and malignant samples. PCA is then performed on the data matrix, comprised of the six spectral features extracted from each of 45 calibration samples, and three principal components (PCs) containing maximum diagnostic information are selected. The scores of the selected PCs are used to train the k-NN, ANN, and SVM classifiers. The ANN used is a classical multilayer feed forward network with back propagation algorithm for its training. For k-NN, the Euclidean distance based algorithm is used and for SVM, one-versus-rest multiclass kernel-radial basis function is used. The performance evaluation of the classification results are obtained by calculating statistical parameters like specificity and sensitivity. ANN and k-NN techniques showed identical performance with specificity and sensitivity values of 100 and 86.76%, whereas SVM had these values at 100 and 80.18%, respectively. In order to determine the relative diagnostic performance of the techniques, receiver operating characteristics analysis is also performed.

Ultrasound microbubbles are contrast agents used for diagnostic ultrasound imaging and as carriers for noninvasive payload delivery. Understanding the acoustic properties of individual microbubble formulations is important for optimizing the ultrasound imaging parameters for improved image contrast and efficient payload delivery. We report here a practical and simple optical tool for direct real-time characterization of ultrasound contrast microbubble dynamics based on light scattering. Fourier transforms of raw linear and nonlinear acoustic oscillations, and microbubble cavitations are directly recorded. Further, the power of this tool is demonstrated by comparing clinically relevant microbubble cycle-to-cycle dynamics and their corresponding Fourier transforms.

A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

Background: The involvement of mitochondria in pathological states, such as neurodegenerative diseases, sepsis, stroke, and cancer, are well documented. Monitoring of nicotinamide adenine dinucleotide (NADH) fluorescence in vivo as an intracellular oxygen indicator was established in 1950 to 1970 by Britton Chance and collaborators. We use a multiparametric monitoring system enabling assessment of tissue vitality. In order to use this technology in clinical practice, the commercial developed device, the CritiView (CRV), is tested in animal models as well as in patients. Methods and Results: The new CRV enables the optical monitoring of four different parameters, representing the energy balance of various tissues in vivo. Mitochondrial NADH is measured by surface fluorometry/reflectometry. In addition, tissue microcirculatory blood flow, tissue reflectance and oxygenation are measured as well. The device is tested both in vitro and in vivo in a small animal model and in preliminary clinical trials in patients undergoing vascular or open heart surgery. In patients, the monitoring is started immediately after the insertion of a three-way Foley catheter (urine collection) to the patient and is stopped when the patient is discharged from the operating room. The results show that monitoring the urethral wall vitality provides information in correlation to the surgical procedure performed.

The aim of this study is to evaluate the biochemical foundation and clinical capability of an image-guided near-infrared (NIR) autofluorescence (AF) spectroscopy technique for in vivo diagnosis of colonic malignancies during clinical colonoscopy. A novel endoscopic fiber-optic AF system was utilized for in vivo NIR AF measurements at 785 nm excitation. A total of 263 in vivo NIR AF spectra of colonic tissues were measured from 100 patients, in which 164 spectra were from benign tissue (116 normal and 48 hyperplastic polyps), 34 spectra were from precancer (adenomatous polyps), and 65 spectra were from cancer. The non-negativity constrained least squares minimization biochemical modeling was explored to estimate the biochemical compositions of colonic tissue using nine basis reference spectra from the representative biochemicals (i.e., collagen I, elastin, β-nicotinamide adenine dinucleotide, flavin adenine dinucleotide, L-tryptophan, hematoporphyrin, 4-pyridoxic acid, pyridoxal 5'-phosphate, and water) associated with structural or cellular metabolic progression in colonic precancer and cancer. High-quality in vivo NIR AF spectra in the spectral range of 810 to 1000 nm were acquired from colonic benign, precancerous, and cancerous mucosa under white-light reflectance endoscopic imaging guidance. Partial least squares discriminant analysis, together with the leave-one tissue site-out, cross validation on in vivo NIR AF spectra yields diagnostic sensitivities of 85.4%, 76.5%, and 84.6%, and specificities of 89.9%, 93.4%, and 91.4%, respectively, for classification of benign, precancer, and cancer in the colon. This work demonstrates that image-guided NIR AF spectroscopy in conjunction with biochemical modeling has promising potential for improving in vivo detection and diagnosis of colonic precancer and cancer during clinical colonoscopic screening.

The human skin is modeled as a stack of homogeneous layers in the terahertz and submillimeter waves regions with some anisotropy due to the helical sweat glands and other elongated entities. A dielectric model for the skin is presented, valid for a wider frequency range (up to the terahertz region) taking into account the dispersive nature of the effective conductivity. Polarized reflectivity and generalized ellipsometric parameters are calculated versus angle and wavelength. Recent studies have claimed that the helical sweat ducts act as an array of low-Q helical antennae and are dominant in shaping the spectral response in the sub-terahertz region. We found that water absorption, dispersion and multiple interference effects play the major role in shaping the spectrum without the need for the assumption of the sweat ducts acting as low-Q helical antennae. High sensitivities to the water content are found particularly in the ellipsometric parameters at large incidence angles. Hence a new methodology is proposed to detect skin cancer using variable angle ellipsometry or polarized reflectometry. The parameter found with the highest sensitivity to water content is cos Δpp with Δpp being the phase of the on-diagonal reflection matrix ratio between p-to-p polarization.

Low-coherence enhanced backscattering (LEBS) is a depth selective technique that allows noninvasive characterization of turbid media such as biological tissue. LEBS provides a spectral measurement of the tissue reflectance distribution as a function of distance between incident and reflected ray pairs through the use of partial spatial coherence broadband illumination. We present LEBS as a new depth-selective technique to measure optical properties of tissue insitu. Because LEBS enables measurements of reflectance due to initial scattering events, LEBS is sensitive to the shape of the phase function in addition to the reduced scattering coefficient (μs*). We introduce a simulation of LEBS that implements a two parameter phase function based on the Whittle-Matérn refractive index correlation function model. We show that the LEBS enhancement factor (E) primarily depends on μs*, the normalized spectral dependence of E (S_n) depends on one of the two parameters of the phase function that also defines the functional type of the refractive index correlation function (m), and the LEBS peak width depends on both the anisotropy factor (g) and m. Three inverse models for calculating these optical properties are described and the calculations are validated with an experimental measurement from a tissue phantom.

Recently, resting-state functional near-infrared spectroscopy (rs-fNIRS) research has experienced tremendous progress. Resting-state functional connectivity (RSFC) has been adopted as a pivotal biomarker in rs-fNIRS studies. However, it is yet to be clear if the RSFC derived from rs-fNIRS is reliable. This concern impedes extensive utilization of rs-fNIRS. We systematically address the issue of reliability. Sixteen subjects participate in two rs-fNIRS sessions held one week apart. RSFC in sensorimotor system is calculated using the seed-correlation approach. Then, test-retest reliability is evaluated at three different scales (map-, cluster-, and channelwise) for individual- and group-level RSFC derived from different types of fNIRS signals [oxygenated (HbO), deoxygenated (HbR), and total hemoglobin (HbT)]. The results show that, for HbO signals, individual-level RSFC generally has good-to-excellent map-/clusterwise reliability, while group-level RSFC has excellent reliability. For HbT signals, the results are similar. For HbR signals, the clusterwise reliability is comparable to that for HbO while the mapwise reliability is slightly lower (fair to good). Focusing on RSFC at a single channel, we report poor channelwise reliability for all three types of signals. We hereby propose that fNIRS-derived RSFC is a reliable biomarker if interpreted in map- and clusterwise manners. However, channelwise interpretation of individual RSFC should proceed with caution.

Optical spectroscopy has shown potential as a real-time, in vivo, diagnostic tool for identifying neoplasia during endoscopy. We present the development of a diagnostic algorithm to classify elastic-scattering spectroscopy (ESS) spectra as either neoplastic or non-neoplastic. The algorithm is based on pattern recognition methods, including ensemble classifiers, in which members of the ensemble are trained on different regions of the ESS spectrum, and misclassification-rejection, where the algorithm identifies and refrains from classifying samples that are at higher risk of being misclassified. These "rejected" samples can be reexamined by simply repositioning the probe to obtain additional optical readings or ultimately by sending the polyp for histopathological assessment, as per standard practice. Prospective validation using separate training and testing sets result in a baseline performance of sensitivity = .83, specificity = .79, using the standard framework of feature extraction (principal component analysis) followed by classification (with linear support vector machines). With the developed algorithm, performance improves to Se~90, Sp~0.90, at a cost of rejecting 20-33% of the samples. These results are on par with a panel of expert pathologists. For colonoscopic prevention of colorectal cancer, our system could reduce biopsy risk and cost, obviate retrieval of non-neoplastic polyps, decrease procedure time, and improve assessment of cancer risk.

We study fluorescence lifetime of indocyanine green (ICG) using femtosecond laser and sensitive detection based on time-correlated single-photon counting. A time-resolved multichannel spectral system is constructed and applied for determination of the fluorescence lifetime of the ICG in different solvents. Emission properties of ICG in water, milk, and 1% intralipid solution are investigated. Fluorescence of the fluorophore of different concentrations (in a range of 1.7-160 μM) dissolved in different solutions is excited by femtosecond pulses generated with the use of Ti:Sa laser tuned within the range of 740-790 nm. It is observed that fluorescence lifetime of ICG in water is 0.166 ± 0.02 ns and does not depend on excitation and emission wavelengths. We also show that for the diffusely scattering solvents (milk and intralipid), the lifetime may depend on the dye concentration (especially for large concentrations of ICG). This effect should be taken into account when analyzing changes in the mean time of arrival of fluorescence photons excited in ICG dissolved in such optically turbid media.

The purpose of the present study was to objectively quantify the spectral transmittance of the eyelid. Reported here are data acquired using a technique that was developed to provide practical and accurate measurements of eyelid transmittance across the visible portion of the electromagnetic spectrum. The empirical data were analyzed in terms of the absorption and scattering characteristics of the constituents of skin to develop a method for predicting eyelid transmission. Results showed that the eyelid has a much higher optical density at short wavelengths than previously published. The mean ± standard deviation (s.d.) optical density of the eyelid from 450 to 650 nm was 2.1 ± 0.3 with an optical density range among subjects of approximately 1.0. The study results indicate that skin pigmentation is poorly correlated with eyelid transmission; eyelid transmission is most affected by wavelength-independent macromolecules in the eyelid as well as its overall thickness.

A major challenge in endocrine surgery is the intraoperative detection of parathyroid glands during both thyroidectomies and parathyroidectomies. Current localization techniques such as ultrasound and sestamibi scan are mostly preoperative and rely on an abnormal parathyroid for its detection. In this paper, we present near-infrared (NIR) autofluorescence as a nonintrusive, real-time, automated in vivo method for the detection of the parathyroid gland. A pilot in vivo study was conducted to assess the ability of NIR fluorescence to identify parathyroid glands during thyroid and parathyroidectomies. Fluorescence measurements at 785 nm excitation were obtained intra-operatively from the different tissues exposed in the neck region in 21 patients undergoing endocrine surgery. The fluorescence intensity of the parathyroid gland was found to be consistently greater than that of the thyroid and all other tissues in the neck of all patients. In particular, parathyroid fluorescence was two to eleven times higher than that of the thyroid tissues with peak fluorescence occurring at 820 to 830 nm. These results indicate that NIR fluorescence has the potential to be an excellent optical tool to locate parathyroid tissue during surgery.

Noninvasive detection of cell death has the potential for definitive diagnosis and monitoring treatment outcomes in real time. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence intensity has long been used as a noninvasive optical probe of metabolic states. NADH fluorescence lifetime has recently been studied for its potential as an alternative optical probe of cellular metabolic states and cell death. In this study, we investigated the potential using NADH fluorescence intensity and/or lifetime to detect poly(adenosine-5'-diphosphate-ribose) polymerase-1 (PARP-1)-mediated cell death in HeLa cells. We also examined if NADH signals respond to treatment by pyruvate. The mechanism of PARP-1-mediated cell death has been well studied that extensive PARP-1 activation leads to cytosolic nicotinamide adenine dinucleotide depletion resulting in glycolytic inhibition, mitochondrial failure, and death. Pyruvate could restore electron transport chain to prevent energy failure and death. Our results show that NADH fluorescence lifetime, not intensity, responded to PARP-1-mediated cell death and the rescue effect of pyruvate. This lifetime change of NADH fluorescence happened before the collapse of mitochondrial membrane potential and mitochondrial uncoupling. Together with our previous findings in staurosporine-induced cell death, we suggest that NADH fluorescence lifetime increase during cell death is mainly due to increased protein-protein interactions but not the intracellular NADH content.

Changes have been made to this article. See the full text for a description of the changes.

Changes have been made to this article. See the full text for a description of the changes.