An algorithm was developed to obtain depth-resolved local optical axis in birefringent samples by using conventional polarization-sensitive optical coherence tomography (PSOCT) that uses a single circularly polarized incident light. The round-trip sample Jones matrices were first constructed from the cumulative PSOCT results. An iterative method was then applied to construct the depth-resolved local Jones matrix from which the local optical axis was calculated. The proposed algorithm was validated in samples with homogeneous axis and with depth-varying optical axis. Imaging examples were shown to demonstrate the capability of this method for extracting correct local axis and revealing features not evident in other images.

Many studies suggest that the degradation of microtubules in the retinal ganglion cells may be an early event in the progression of glaucoma. Because reflectance and birefringence of the retinal nerve fibers arise primarily from microtubules, the optical properties have been intensively studied for early detection of the disease. We previously reported a novel nonlinear optical signal from axonal microtubules for visualizing the retinal nerve fibers, namely second-harmonic generation (SHG). We demonstrate the use of axonal SHG to investigate the effect of microtubules on the morphology of the retinal nerve fiber bundles. Time-lapse SHG imaging of ex vivo rat retinal flat mounts was performed during pharmacological treatment of nocodazole, and the intensity of axonal SHG and the changes in nerve fiber bundle morphology were monitored. We found that the microtubule disruption does not lead to immediate modification in the morphology of the nerve fibers. Our results indicate that microtubular SHG may provide a useful means for sensitive detection of axonal injuries. Since the intrinsic radiation depends on the regular architecture of the cytoskeleton element as maintained by active cellular regulations, the intensity of signal reflects the health of the retinal ganglion cell axons.

Photoacoustic (PA) imaging was applied to detect the neuronal activity in the motor cortex of an awake, behaving monkey during forelimb movement. An adult macaque monkey was trained to perform a reach-to-grasp task while PA images were acquired through a 30-mm diameter implanted cranial chamber. Increased PA signal amplitude results from an increase in regional blood volume and is interpreted as increased neuronal activity. Additionally, depth-resolved PA signals enabled the study of functional responses in deep cortical areas. The results demonstrate the feasibility of utilizing PA imaging for studies of functional activation of cerebral cortex in awake monkeys performing behavioral tasks.

The quality of optoacoustic tomographic reconstructions can be severely affected by acoustic reflections or scattering arising at interfaces of highly mismatched organs, such as bones, lungs, or other air-containing cavities. We present a procedure to reduce the associated artefacts based on estimation of the acoustic scatterers distribution within the imaged object. Signals generated by a strong optical absorber are processed and used in a weighted back-projection algorithm. Experimental results in a tissue-mimicking phantom clearly demonstrate improved performance as compared to the case in which no information on the distribution of acoustic scatterers is available.

Many diseases involve changes in the biomechanical properties of tissue, and there is a close correlation between tissue elasticity and pathology. We report on the development of a phase-resolved acoustic radiation force optical coherence elastography method (ARF-OCE) to evaluate the elastic properties of tissue. This method utilizes chirped acoustic radiation force to produce excitation along the sample's axial direction, and it uses phase-resolved optical coherence tomography (OCT) to measure the vibration of the sample. Under 500-Hz square wave modulated ARF signal excitation, phase change maps of tissue mimicking phantoms are generated by the ARF-OCE method, and the resulting Young's modulus ratio is correlated with a standard compression test. The results verify that this technique could efficiently measure sample elastic properties accurately and quantitatively. Furthermore, a three-dimensional ARF-OCE image of the human atherosclerotic coronary artery is obtained. The result indicates that our dynamic phase-resolved ARF-OCE method can delineate tissues with different mechanical properties.

Photoacoustic tomography (PAT) of the human brain is challenging due to the fact that the skull strongly absorbs and scatters light, and attenuates and distorts ultrasound as well. For the first time, we demonstrated the feasibility of PAT through a whole adult human skull. A photon recycler (PR) was built to increase light transmittance through the skull. Both a graphite target and a canine brain were imaged through the skull. Use of the PR was found to improve the photoacoustic signal-to-noise ratio by a factor of 2.4. In addition, subtraction of photoacoustic signals that arise from light absorption within the skull significantly improved the contrast of the target. Our results indicate that PAT can potentially be applied to in vivo human brain imaging.

Obstruction in the upper airway can often cause reductions in breathing or gas exchange efficiency and lead to rest disorders such as sleep apnea. Imaging diagnosis of the obstruction region has been accomplished using computed tomography (CT) and magnetic resonance imaging (MRI). However CT requires the use of ionizing radiation, and MRI typically requires sedation of the patient to prevent motion artifacts. Long-range optical coherence tomography (OCT) has the potential to provide high-speed three-dimensional tomographic images with high resolution and without the use of ionizing radiation. In this paper, we present work on the development of a long-range OCT endoscopic probe with 1.2 mm OD and 20 mm working distance used in conjunction with a modified Fourier domain swept source OCT system to acquire structural and anatomical datasets of the human airway. Imaging from the bottom of the larynx to the end of the nasal cavity is completed within 40 s.

A survey of the literature is presented regarding the simulation of port wine stain (PWS) skin color. Knowledge of PWS features, such as the depths and diameters of affected vessels, is essential for informing laser treatment. These may be determined through the inverse application of a skin model. The techniques which have been applied to achieve this are analyzed in detail. Radiative transfer (RT) is found to be the preferred method of simulation. By far the most common approximations to RT are the diffusion approximations, which have been applied successfully in the past and Monte Carlo techniques, which are now the methods of choice. As the requirements for improvement of laser treatment on an individual basis continues, the needs for further work towards accurate estimations of individual optical coefficients and robust, flexible simulation techniques are identified.

We present an open source electric field tracking Monte Carlo program to model backscattering in biological media containing birefringence, with computation of the coherent backscattering phenomenon as an example. These simulations enable the modeling of tissue scattering as a statistically homogeneous continuous random media under the Whittle-Matérn model, which includes the Henyey-Greenstein phase function as a special case, or as a composition of discrete spherical scatterers under Mie theory. The calculation of the amplitude scattering matrix for the above two cases as well as the implementation of birefringence using the Jones N-matrix formalism is presented. For ease of operator use and data processing, our simulation incorporates a graphical user interface written in MATLAB to interact with the underlying C code. Additionally, an increase in computational speed is achieved through implementation of message passing interface and the semi-analytical approach. Finally, we provide demonstrations of the results of our simulation for purely scattering media and scattering media containing linear birefringence.

Because direct measurements of the refractive index of hemoglobin over a large wavelength range are challenging, indirect methods deserve particular attention. Among them, the Kramers-Kronig relations are a powerful tool often used to derive the real part of a refractive index from its imaginary part. However, previous attempts to apply the relations to solutions of human hemoglobin have been somewhat controversial, resulting in disagreement between several studies. We show that this controversy can be resolved when careful attention is paid not only to the absorption of hemoglobin but also to the dispersion of the refractive index of the nonabsorbing solvent. We present a Kramers-Kroning analysis taking both contributions into account and compare the results with the data from several studies. Good agreement with experiments is found across the visible and parts of near-infrared and ultraviolet regions. These results reinstate the use of the Kramers-Kronig relations for hemoglobin solutions and provide an additional source of information about their refractive index.

So far, the study of the optical clearing is almost always about healthy tissue. However, the ultimate goal is to detect diseases for clinical application. Optical clearing on diseased skins is explored. The effect is evaluated by applying a combined liquid paraffin and glycerol mixed solution on several kinds of diseased skins in vitro. Scanning experiments from optical coherence tomography show that it has different effects among fibroma, pigmented nevus, and seborrheic keratosis. Based on the results, we conclude that different skin diseases have different compositions and structures, and their optical parameters and biological characteristics should be different, which implies that the optical clearing technique may have selectivity and may not be suitable for all kinds of skin diseases.

The Z-Scan (ZS) technique in the thermal regime has been used to measure the nonlinear optical response of low-density lipoprotein (LDL). The ZS technique is carried out in LDL from 40 patients with chronic periodontitis before and after three, six, and 12 months of periodontal treatment. Clinical parameters such as probing depths, bleeding on probing, total and differential white blood cells counts, lipid profiles, cytokine levels, and antibodies against oxidized LDL are also determined and compared over time. Before the treatment, the ZS experimental results reveal that the LDL particles of these patients are heavily modified. Only after 12 months of the periodontal treatment, the ZS results obtained reveal behavioral characteristics of healthy particles. This conclusion is also supported by complementary laboratorial analysis showing that the periodontal treatment induces systemic changes in several inflammatory markers.

Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F₆₄PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F₆₄PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers' oxidation as well as O₂ saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.

Kubelka-Munk (K-M) theory is a phenomenological light transport theory that provides analytical expressions for reflectance and transmittance of diffusive substrates such as tissues. Many authors have derived relations between coefficients of K-M theory and that of the more fundamental radiative transfer equations. These relations are valid only in diffusive light transport regime where scattering dominates over absorption. They also fail near boundaries where incident beams are not diffusive. By measuring total transmittance and total reflectance of tissue phantoms with varying optical parameters, we have obtained empirical relations between K-M coefficients and the radiative transport coefficients for integrating sphere-based spectrophotometers that use uniform, nondiffusive incident beams. Our empirical relations show that the K-M scattering coefficients depend only on reduced scattering coefficient (μ′_s), whereas the K-M absorption coefficient depends on both absorption (μ_a) and reduced scattering (μ′_s) coefficients of radiative transfer theory. We have shown that these empirical relations are valid in both the diffusive and nondiffusive regimes and can predict total reflectance within an error of 10%. They also can be used to solve the inverse problem of obtaining multiple optical parameters such as chromophore concentration and tissue thickness from the measured reflectance spectra with a maximum accuracy of 90% to 95%.

Tissular oxygen concentration plays a key role during photodynamic therapy (PDT). Therefore, monitoring its local oxygen partial pressure (pO₂) may help predict and/or control the outcome of a PDT treatment. The first real-time, in vivo measurements of the pO₂ in the chicken egg's chorioallantoic membrane, using the delayed fluorescence of photoactivable porphyrins (PAPs), including protoporphyrin IX (PpIX), as monitored with a dedicated optical, fiber-based, time-resolved spectrometer, are reported here. The formation of PAPs/PpIX, photosensitizers of extensive clinical use, was induced in the chicken egg's chorioallantoic membrane (CAM) with aminolevulinic acid. An excellent correlation between the vascular damage induced by PDT and the reduction in tissular pO₂ is found. This study suggests that clinical measurement of the pO₂ using the PAPs'/PpIX's delayed fluorescence (DF) may be used to individualize in real time the PDT light dose applied.

Fluorescence microscopy offers high sensitivity for disease diagnosis. However, little structural information is revealed by this method, requiring another technique to localize the source of fluorescence. We developed a complementary fluorescence-polarization microscope. We used a division-of-focal-plane charge coupled device polarization sensor to enable real-time video rate polarization imaging without any moving parts. The polarization information provided by the microscope enabled detection of structural element and complements the fluorescence information. Application of this multimodal system for cancer imaging using a tumor selective molecular probe revealed the association of diminished structural integrity of tumor tissue with high fluorescence of the imaging agent compared to surrounding normal tissue. This study demonstrates a new paradigm to improve cancer detection and diagnosis.

We evaluated preclinical single-dose safety, pharmacokinetic properties, and specific uptake of the new optical imaging agent fluorocoxib A in dogs. Fluorocoxib A, N-[(5-carboxy-X-rhodaminyl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide, selectively binds and inhibits the cyclooxygenase-2 (COX-2) enzyme, which is overexpressed in many cancers. Safety pilot studies were performed in research dogs following intravenous (i.v.) administration of 0.1 and 1  mg/kg fluorocoxib A. Blood and urine samples collected three days after administration of each dose of fluorocoxib A revealed no evidence of toxicity, and no clinically relevant adverse events were noted on physical examination of exposed dogs over that time period. Pharmacokinetic parameters were assessed in additional research dogs from plasma collected at several time points after i.v. administration of fluorocoxib A using high-performance liquid chromatography analysis. The pharmacokinetic studies using 1  mg/kg showed a peak of fluorocoxib A (92±28  ng) in plasma collected at 0.5 h. Tumor specific uptake of fluorocoxib A was demonstrated using a dog diagnosed with colorectal cancer expressing COX-2. Our data support the safe single-dose administration and in vivo efficacy of fluorocoxib A, suggesting a high potential for successful translation to clinical use as an imaging agent for improved tumor detection in humans.

Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-μm axial, 65-μmm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins.

We have explored the use of optical coherence tomography (OCT) as a noninvasive tool for assessing the toxicity of topical microbicides, products used to prevent HIV, by monitoring the integrity of the vaginal epithelium. A novel feature-based segmentation algorithm using a nearest-neighbor classifier was developed to monitor changes in the morphology of vaginal epithelium. The two-step automated algorithm yielded OCT images with a clearly defined epithelial layer, enabling differentiation of normal and damaged tissue. The algorithm was robust in that it was able to discriminate the epithelial layer from underlying stroma as well as residual microbicide product on the surface. This segmentation technique for OCT images has the potential to be readily adaptable to the clinical setting for noninvasively defining the boundaries of the epithelium, enabling quantifiable assessment of microbicide-induced damage in vaginal tissue.

To evaluate retinal perfusion in the human eye, a dual-wavelength confocal scanning laser ophthalmoscope (cSLO) was developed that provides spectral imaging of the fundus using a combination of red (670 nm) and near-infrared (810 nm) wavelengths. The image of the ocular fundus was analyzed to find out if quantitative measurements of the reflectivity of tissue permit assessment of the oxygen perfusion of tissue. We explored problems that affect the reproducibility of patient measurements such as non-uniformity errors on the image. For the first time, an image processing technique was designed and used to minimize the errors of oxygen saturation measurements by illumination correction in retina wide field by increasing SNR. Retinal images were taken from healthy and diabetic retinopathy eyes using the cSLO with a confocal aperture of 100 μm. The ratio image (RI) of red/IR, as oxygen saturation (SO₂) index, was calculated for normal eyes. The image correction technique improved the reproducibility of the measurements. Average RI intensity variation of healthy retina tissue was determined within a range of about 5.5%. The capability of the new technique to discriminate oxygenation levels of retinal artery and vein was successfully demonstrated and showed good promise in the diagnosis of the perfused retina.

The objective of this study was to evaluate the effects of ultrasound-mediated analyte diffusion on permeability of normal, benign, and cancerous human lung tissue in vitro and to find more effective sonophoretic (SP) delivery in combination with the optical clearing agents (OCAs) method to distinguish normal and diseased lung tissues. The permeability coefficients of SP in combination with OCAs diffusion in lung tissue were measured with Fourier-domain optical coherence tomography (FD-OCT). 30% glucose and SP with a frequency of 1 MHz and an intensity of 0.80 W/cm² over a 3 cm probe was simultaneously applied for 15 min. Experimental results show that the mean permeability coefficients of 30% glucose/SP were found to be (2.01±0.21)×10^-5 cm/s from normal lung (NL) tissue, (2.75±0.28)×10^-5 cm/s from lung benign granulomatosis (LBG) tissue, (4.53±0.49)×10^-5 cm/s from lung adenocarcinoma tumor (LAT) tissue, and (5.81±0.62)×10^-5 cm/s from lung squamous cell carcinoma (LSCC) tissue, respectively. The permeability coefficients of 30% glucose/SP increase approximately 36.8%, 125.4%, and 189.1% for the LBG, LAT, and LSCC tissue compared with that for the NL tissue, respectively. There were statistically significant differences in permeability coefficients of 30% glucose/SP between LBG and NL tissue (p<0.05), between LAT and NL tissue (p<0.05), and between LSCC and NL tissue (p<0.05). The results suggest that the OCT functional imaging technique to combine an ultrasound-OCAs combination method could become a powerful tool in early diagnosis and monitoring of changed microstructure of pathologic human lung tissue.

Atopic dermatitis (AD) is an allergic inflammatory disease of skin. It remains unclear that CD207 of Langerhans cells (LCs) plays a central role in the development of allergic sensitization. There is little data on LCs within the microenviroment in vivo. We used a murine model of epicutaneous (EC) ovalbumin (OVA) sensitization inducing an inflammatory skin resembling AD to explore the role of CD207 in the pathogenesis of AD. Cr:forsterite laser-based multimodality nonlinear microscopy was applied for in situ imaging. Peritoneal injections of Alexa Fluor 647-rat anti-mouse CD207 into mice were performed to specifically trace the LCs. Peritoneal injections of OVA-Alexa Fluor 647 conjugate into mice were performed to specifically trace the OVA. We found that combining Alexa Fluor fluorescent probes with multimodality nonlinear microscopy permitted the unequivocal in situ imaging of CD207-expressing LCs. The relevant time-course, expressional, and functional studies reveal that CD207 of LCs plays an essential role during the induction of EC sensitization. We establish and validate that Cr:forsterite laser-based multimodality nonlinear microscopy is applicable for the specific detection of labeled mAb-bound LCs and labeled antigen. We suggest that CD207-expressing LCs initiate the allergic response through the CD207 mediated epicutaneous sensitization associated with the development of AD.

Thirty-seven normal and primary open angle glaucoma (POAG) subjects were noninvasively imaged by a tailor-made real-time anterior segment swept source optical coherence tomography (SS-OCT) to demonstrate the differences of the Schlemm's canal (SC) between POAG and normal eyes. After the cross-section images of the anterior chamber angle were acquired by SS-OCT, SC was confirmed by two independent masked observers and the average area, long diameter, and perimeter of the SC were measured. In normal subjects the circumference, area, and long diameter is <b<508.34±87.81 μm</b<, <b<8023.89±1486.10  μm²</b<, and <b<272.83±49.39  μm</b<, respectively, and these parameters were <b<393.25±98.04  μm</b<, <b<3941.50±1210.69  μm²</b<, and <b<190.91±46.47 μm</b<  in the POAG subjects. The area of SC in the normal ones was significantly larger than that in POAG eyes (<b<p<0.001</b<), so as the long diameter and the perimeter (<b<p<0.001</b<; <b<p<0.001</b<).

Image enhancement of retinal structures, in optical coherence tomography (OCT) scans through denoising, has the potential to aid in the diagnosis of several eye diseases. In this paper, a locally adaptive denoising algorithm using double-density dual-tree complex wavelet transform, a combination of the double-density wavelet transform and the dual-tree complex wavelet transform, is applied to reduce speckle noise in OCT images of the retina. The algorithm overcomes the limitations of commonly used multiple frame averaging technique, namely the limited number of frames that can be recorded due to eye movements, by providing a comparable image quality in significantly less acquisition time equal to an order of magnitude less time compared to the averaging method. In addition, improvements of image quality metrics and 5 dB increase in the signal-to-noise ratio are attained.

A custom-developed ultrahigh resolution optical coherence tomography with an axial resolution of 1.1 μm in corneal tissue was used to characterize thickness and light scatter of the epithelium and Bowman's layer in keratoconic (KC) cornea noninvasively. A 4-mm wide vertical corneal section around the apex in nine KC and eight normal eyes was imaged in vivo. The epithelium and Bowman's layer were visualized and their thickness profiles were quantified. Scatter was quantified based on the sensitivity normalized mean signal intensity distribution. Average mean thickness of the epithelium and Bowman's layer in KC eyes was significantly smaller (<b<p<0.05</b<) than the normal eyes. The epithelium thickness variation across a central 3-mm cornea was significantly larger in KC eyes than in normal eyes. The scatter in KC eyes was significantly increased only for Bowman's layer. The changes observed in this study could improve our understanding of the underlying disease mechanism of KC and can provide new indications for early disease diagnosis.

Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

Innovative diagnostic methods are the need of the hour that could complement conventional histopathology for cancer diagnosis. In this perspective, we propose a new concept based on spectral histopathology, using IR spectral micro-imaging, directly applied to paraffinized colon tissue array stabilized in an agarose matrix without any chemical pre-treatment. In order to correct spectral interferences from paraffin and agarose, a mathematical procedure is implemented. The corrected spectral images are then processed by a multivariate clustering method to automatically recover, on the basis of their intrinsic molecular composition, the main histological classes of the normal and the tumoral colon tissue. The spectral signatures from different histological classes of the colonic tissues are analyzed using statistical methods (Kruskal-Wallis test and principal component analysis) to identify the most discriminant IR features. These features allow characterizing some of the biomolecular alterations associated with malignancy. Thus, via a single analysis, in a label-free and nondestructive manner, main changes associated with nucleotide, carbohydrates, and collagen features can be identified simultaneously between the compared normal and the cancerous tissues. The present study demonstrates the potential of IR spectral imaging as a complementary modern tool, to conventional histopathology, for an objective cancer diagnosis directly from paraffin-embedded tissue arrays.

We used a probe-based dual-modality optical imaging instrument to measure in vivo coating thickness distributions of a gel distributed along the vaginal lumen, in a clinical study. The gel was a surrogate for one delivering an anti-HIV topical microbicide. Imaging data from Fourier-domain multiplexed low-coherence interferometry (mLCI) and fluorimetric measurements were compared to assess the feasibility and accuracy of mLCI in measuring in vivo gel coating thickness distributions. In each study session, 3.5 mL of Replens gel was inserted to the vaginal fornix while the participant was supine. The participant either: 1. remained supine (10 or 60 min); or 2. sat up (1 min), stood up (1 min), sat down (1 min) and returned to the supine position; net elapsed time was 10 or 60 min after which the gel distribution was imaged. Local coating thickness distributions were qualitatively and quantitatively similar. Here mLCI did not accurately measure thicker gel coatings (<b<<0.8 mm</b<), a limitation not seen with fluorimetry. However, mLCI is capable of measuring in vivo microbicide gel distributions with resolution on the order of 10 μm, without the need for exogenous contrast agents, and can accurately capture relevant summary coating measures in good agreement with fluorimetry.

Optical measurements of metabolism are ideally acquired in vivo; however, intravital measurements are often impractical. Accurate ex vivo assessments would greatly broaden the applicability of optical measurements of metabolism. We investigate the use of live tissue culture experiments to serve as a surrogate for in vivo metabolic measurements. To validate this approach, NADH and FAD fluorescence intensity and lifetime images were acquired with a two-photon microscope from hamster cheek pouch epithelia in vivo, from biopsies maintained in live tissue culture up to 48 h, and from flash-frozen and thawed biopsies. We found that the optical redox ratio (fluorescence intensity of NADH/FAD) of the cultured biopsy was statistically identical to the in vivo measurement until 24 h, while the redox ratio of the frozen-thawed samples decreased by 15% (<b<p<0.01</b<). The NADH mean fluorescence lifetime (τ_m) remained unchanged (<b<p<0.05</b<) during the first 8 h of tissue culture, while the NADHτ_m of frozen-thawed samples increased by 13% (<b<p<0.001</b<). Cellular morphology did not significantly change between in vivo, cultured, and frozen-thawed tissues (<b<p<0.05</b<). All results were consistent across multiple depth layers in this stratified squamous epithelial tissue. Histological markers for proliferation and apoptosis also confirm the viability of tissues maintained in culture. This study suggests that short-term ex vivo tissue culture may be more appropriate than frozen-thawed tissue for optical metabolic and morphologic measurements that approximate in vivo status.

Photoacoustic microscopy has achieved submicron lateral resolution, but its axial resolution is much lower. Here an axial resolution of 7.6 μm, the highest axial resolution validated by experimental data, has been achieved by using a commercial 125 MHz ultrasonic transducer for signal detection followed by the Wiener deconvolution for signal processing. Limited by the working distance, the high-frequency ultrasonic transducer can penetrate 1.2 mm into biological tissue from the ultrasound detection side. At this depth, the signal-to-noise ratio decreases by 11 dB, and the axial resolution degrades by 36%. The new system was demonstrated in imaging melanoma cells ex vivo and mouse ears in vivo.

Collagen I (Col1) fibers are a major structural component in the extracellular matrix of human breast cancers. In a preliminary pilot study, we explored the link between Col1 fiber density in primary human breast cancers and the occurrence of lymph node metastasis. Col1 fibers were detected by second harmonic generation (SHG) microscopy in primary human breast cancers from patients presenting with lymph node metastasis (LN⁺) versus those without lymph node metastasis (LN^-). Col1 fiber density, which was quantified using our in-house SHG image analysis software, was significantly higher in the primary human breast cancers of LN⁺ (fiber volume=29.22%±4.72%, inter-fiber distance=2.25±0.45 μm) versus LN^- (fiber volume=20.33%±5.56%, inter-fiber distance=2.88±1.07 μm) patients. Texture analysis by evaluating the co-occurrence matrix and the Fourier transform of the Col1 fibers proved to be significantly different for the parameters of co-relation and energy, as well as aspect ratio and eccentricity, for LN⁺ versus LN^- cases. We also demonstrated that tissue fixation and paraffin embedding had negligible effect on SHG Col1 fiber detection and quantification. High Col1 fiber density in primary breast tumors is associated with breast cancer metastasis and may serve as an imaging biomarker of metastasis.

It is demonstrated that optical microangiography (OMAG) is capable of imaging the detailed microstructure and microvasculature of the in vivo human optic nerve head (ONH), including the prelaminar tissue, the lamina cribrosa, the scleral rim and the vessels in the region of the circle of Zin-Haller. For demonstration, an ultrahigh sensitive OMAG system operating in the 850 nm wavelength region and a 500 kHz A-scan rate resulting in a spatial resolution of ∼6  μm were used. It was shown that OMAG provides superior results for three-dimensional imaging of the ONH compared to conventional optical coherence tomography by simultaneously recording both the microstructure and the functional microcirculation. The blood supply to the tissues of the ONH is an essential physiologic parameter needed for clinical assessment of the health of the nerve.

Achieving real-time photoacoustic (PA) tomography typically requires multi-element ultrasound transducer arrays and their associated multiple data acquisition (DAQ) electronics to receive PA waves simultaneously. We report the first demonstration of a photoacoustic tomography (PAT) system using optical fiber-based parallel acoustic delay lines (PADLs). By employing PADLs to introduce specific time delays, the PA signals (on the order of a few micro seconds) can be forced to arrive at the ultrasonic transducers at different times. As a result, time-delayed PA signals in multiple channels can be ultimately received and processed in a serial manner with a single-element transducer, followed by single-channel DAQ electronics. Our results show that an optically absorbing target in an optically scattering medium can be photoacoustically imaged using the newly developed PADL-based PAT system. Potentially, this approach could be adopted to significantly reduce the complexity and cost of ultrasonic array receiver systems.

Repetitive transcranial magnetic stimulation (rTMS) is a technology that stimulates neurons with rapidly changing magnetic pulses with demonstrated therapeutic applications for various neuropsychiatric disorders. Functional near-infrared spectroscopy (fNIRS) is a suitable tool to assess rTMS-evoked brain responses without interference from the magnetic or electric fields generated by the TMS coil. We have previously reported a channel-wise study of combined rTMS/fNIRS on the motor and prefrontal cortices, showing a robust decrease of oxygenated hemoglobin concentration (Δ[HbO2]) at the sites of 1-Hz rTMS and the contralateral brain regions. However, the reliability of this putative clinical tool is unknown. In this study, we develop a rapid optical topography approach to spatially characterize the rTMS-evoked hemodynamic responses on a standard brain atlas. A hemispherical approximation of the brain is employed to convert the three-dimensional topography on the complex brain surface to a two-dimensional topography in the spherical coordinate system. The test-retest reliability of the combined rTMS/fNIRS is assessed using repeated measurements performed two to three days apart. The results demonstrate that the Δ[HbO2] amplitudes have moderate-to-high reliability at the group level; and the spatial patterns of the topographic images have high reproducibility in size and a moderate degree of overlap at the individual level.

The resonance Raman (RR) spectra of six types of human brain tissues are examined using a confocal micro-Raman system with 532-nm excitation in vitro. Forty-three RR spectra from seven subjects are investigated. The spectral peaks from malignant meningioma, stage III (cancer), benign meningioma (benign), normal meningeal tissues (normal), glioblastoma multiforme grade IV (cancer), acoustic neuroma (benign), and pituitary adenoma (benign) are analyzed. Using a 532-nm excitation, the resonance-enhanced peak at 1548cm-1 (amide II) is observed in all of the tissue specimens, but is not observed in the spectra collected using the nonresonance Raman system. An increase in the intensity ratio of 1587 to 1605cm-1 is observed in the RR spectra collected from meningeal cancer tissue as compared with the spectra collected from the benign and normal meningeal tissue. The peak around 1732cm-1 attributed to fatty acids (lipids) are diminished in the spectra collected from the meningeal cancer tumors as compared with the spectra from normal and benign tissues. The characteristic band of spectral peaks observed between 2800 and 3100cm-1 are attributed to the vibrations of methyl (-CH3) and methylene (-CH2-) groups. The ratio of the intensities of the spectral peaks of 2935 to 2880cm-1 from the meningeal cancer tissues is found to be lower in comparison with that of the spectral peaks from normal, and benign tissues, which may be used as a distinct marker for distinguishing cancerous tissues from normal meningeal tissues. The statistical methods of principal component analysis and the support vector machine are used to analyze the RR spectral data collected from meningeal tissues, yielding a diagnostic sensitivity of 90.9% and specificity of 100% when two principal components are used.

Vulvar squamous cell carcinoma (VSCC) is a gynecological cancer with an incidence of two to three per 100,000 women. VSCC arises from vulvar intraepithelial neoplasia (VIN), which is diagnosed through painful punch biopsy. In this study, optical coherence tomography (OCT) is used to differentiate between normal and VIN tissue. We hypothesize that (a) epidermal layer thickness measured in OCT images is different in normal tissue and VIN, and (b) quantitative analysis of the attenuation coefficient (μ_oct) extracted from OCT data differentiates VIN from normal vulvar tissue. Twenty lesions from 16 patients are imaged with OCT. Directly after data acquisition, a biopsy is performed. Epidermal thickness is measured and values of μ_oct are extracted from 200 OCT scans of normal and VIN tissue. For both methods, statistical analysis is performed using Paired Mann-Whitney-test. Correlation between the two methods is tested using a Spearman-correlation test. Both epidermal layer thickness as well as the μ_oct are different between normal vulvar tissue and VIN lesions (p<0.0001). Moreover, no correlation is found between the epidermal layer thickness and μ_oct. This study demonstrates that both the epidermal thickness and the attenuation coefficient of vulvar epithelial tissue containing VIN are different from that of normal vulvar tissue.

We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing two- and three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.

We encapsulate indocyanine green (ICG) in poly[(D,L-lactide-co-glycolide)-co-PEG] diblock (PLGA-PEG) microballoons for real-time fluorescence and hyperspectral imaging of biliary anatomy. ICG-loaded microballoons show superior fluorescence characteristics and slower degradation in comparison with pure ICG. The use of ICG-loaded microballoons in biliary imaging is demonstrated in both biliary-simulating phantoms and an ex vivo tissue model. The biliary-simulating phantoms are prepared by embedding ICG-loaded microballoons in agar gel and imaged by a fluorescence imaging module in a Da Vinci surgical robot. The ex vivo model consists of liver, gallbladder, common bile duct, and part of the duodenum freshly dissected from a domestic swine. After ICG-loaded microballoons are injected into the gallbladder, the biliary structure is imaged by both hyperspectral and fluorescence imaging modalities. Advanced spectral analysis and image processing algorithms are developed to classify the tissue types and identify the biliary anatomy. While fluorescence imaging provides dynamic information of movement and flow in the surgical region of interest, data from hyperspectral imaging allow for rapid identification of the bile duct and safe exclusion of any contaminant fluorescence from tissue not part of the biliary anatomy. Our experiments demonstrate the technical feasibility of using ICG-loaded microballoons for biliary imaging in

This is the first study of dentin-enamel zone (DEZ) identification with tooth structure characterization based on the optical Stokes-Mueller measurement. Stokes vectors of a cross-sectional tooth slice were measured using various polarization inputs. The direction of the DEZ is different in enamel and dentin structures; therefore, the Stokes profiles can specifically characterize the structures based on the DEZ. This optical method, using polarimetry, provides a useful tool for characterizing tooth.

The hydrodynamic rebalancing laser (HRL) procedure is an ophthalmic therapy based on the administration of subthreshold infrared (810 nm) laser light to selected areas on the retina to treat various retina diseases. Heterogeneities of tissue response are observed, including undesired retinal damages. Variations of tissue absorbance were hypothesized to cause this uneven response. Irradiation parameters (diameter=100  μm; power=1  W; irradiation time: 50 to 200 ms), location and tissue response were studied in 16 patients (20 eyes, 2535 laser spots) to discover any correlation between tissue response and normalized fundus reflectance at 810 nm. The results demonstrate a complex relationship between some pathologies and occurrences of retinal damage, but no clear correlation. One possible reason is that the resolution of reflectance images is insufficient to see "small" (40 μm or less) absorption centers, particularly deep-seated ones. Additionally, tissue parameters other than variations of the fundus optical absorption influence heat diffusion and temperature increases. Monitoring or individualizing the light dose in HRL therapy, or any similar infrared diode laser-based therapy will require more sophisticated technologies, including imaging the retina's reflectance with an improved resolution, as well as refined methods to detect complex correlations between retinal damage and specific pathologies.

Dynamic indocyanine green imaging uses a scanning laser ophthalmoscope and a fluorescent dye to produce movies of the dye-filling pattern in the retina and choroid of the eye. It is used for evaluating choroidal neovascularization. Movies are examined to identify the anatomy of the pathology for planning treatment and to evaluate progression or response to treatment. The popularity of this approach is affected by the complexity and difficulty in interpreting the movies. Software algorithms were developed to produce images from the movies that are easy to interpret. A mathematical model is formulated of the flow dynamics, and a fitting algorithm is designed that solves for the flow parameters. The images provide information about flow and perfusion, including regions of change between examinations. Imaged measures include the dye fill-time, temporal dispersion, and magnitude of the dye dilution temporal curves associated with image pixels. Cases show how the software can help to identify clinically relevant anatomy such as feeder vessels, drain vessels, capillary networks, and normal choroidal draining vessels. As a potential tool for research into the character of neovascular conditions and treatments, it reveals the flow dynamics and character of the lesion. Future varieties of this methodology may be used for evaluating the success of engineered tissue transplants, surgical flaps, reconstructive surgery, breast surgery, and many other surgical applications where flow, perfusion, and vascularity of tissue are important.

An array of in-line curvature sensors on a garment is used to monitor the thoracic and abdominal movements of a human during respiration. The results are used to obtain volumetric changes of the human torso in agreement with a spirometer used simultaneously at the mouth. The array of 40 in-line fiber Bragg gratings is used to produce 20 curvature sensors at different locations, each sensor consisting of two fiber Bragg gratings. The 20 curvature sensors and adjoining fiber are encapsulated into a low-temperature-cured synthetic silicone. The sensors are wavelength interrogated by a commercially available system from Moog Insensys, and the wavelength changes are calibrated to recover curvature. A three-dimensional algorithm is used to generate shape changes during respiration that allow the measurement of absolute volume changes at various sections of the torso. It is shown that the sensing scheme yields a volumetric error of 6%. Comparing the volume data obtained from the spirometer with the volume estimated with the synchronous data from the shape-sensing array yielded a correlation value 0.86 with a Pearson's correlation coefficient p<0.01.

Pain is a natural alarm that aids the body in avoiding potential danger and can also present as an important indicator in clinics. Infrared laser-evoked potentials can be used as an objective index to evaluate nociception. In animal studies, a short-pulse laser is crucial because it completes the stimulation before escape behavior. The objective of the present study was to obtain the temporal and spatial temperature distributions in the skin caused by the irradiation of a short-pulse laser. A fast speed infrared camera was used to measure the surface temperature caused by a CO2 laser of different durations (25 and 35 ms) and power. The measured results were subsequently implemented with a three-layer finite element model to predict the subsurface temperature. We found that stratum corneum was crucial in the modeling of fast temperature response, and escape behaviors correlated with predictions of temperature at subsurface. Results indicated that the onset latency and duration of activated nociceptors must be carefully considered when interpreting physiological responses evoked by infrared irradiation.

Cerebral Autoregulation, in clinical practice, is assessed by means of correlation or coherence analysis between mean arterial blood pressure (MABP) and cerebral blood flow (CBF). However, even though there is evidence linking cerebral autoregulation assessment with clinical outcome in preterm infants, available methods lack precision for clinical use. Classical methods, used for cerebral autoregulation, are influenced by the choice of parameters such as the length of the epoch under analysis and the choice of suitable frequency bands. The influence of these parameters, in the derived measurements for cerebral autoregulation, has not yet been evaluated. In this study, cerebral autoregulation was assessed using correlation, coherence, a modified version of coherence and transfer function gain, and phase. The influence of the extra-parameters on the final scores was evaluated by means of sensitivity analysis. The methods were applied to a database of 18 neonates with measurements of MABP and tissue oxygenation index (TOI). TOI reflects changes in CBF and was measured by means of near-infrared spectroscopy.

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets ∝νβ₃ cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

The complex technique of concerted polarization-phase and spatial-frequency filtering of blood plasma laser images is suggested. The possibility of obtaining the coordinate distributions of phases of linearly and circularly birefringent protein networks of blood plasma separately is presented. The statistical (moments of the first to fourth orders) and scale self-similar (logarithmic dependences of power spectra) structure of phase maps of different types of birefringence of blood plasma of two groups of patients-healthy people (donors) and those suffering from rectal cancer-is investigated. The diagnostically sensitive parameters of a pathological change of the birefringence of blood plasma polycrystalline networks are determined. The effectiveness of this technique for detecting change in birefringence in the smears of other biological fluids in diagnosing the appearance of cholelithiasis (bile), operative differentiation of the acute and gangrenous appendicitis (exudate), and differentiation of inflammatory diseases of joints (synovial fluid) is shown.

A darkfield scattering microscope has been constructed that enables both angular and spectrally resolved measurements of elastic scattering patterns. The comparison of the angular and spectral resolution modes is shown in detail. Angular patterns of the backscattered light by homogeneous polystyrene spheres were measured at 57 wavelengths and the diameters of the single spheres were determined by using Mie theory at each wavelength. The mean diameter values were estimated in the angular mode with a relative standard deviation of 0.25% or less. Spectral scattering patterns of the same beads were investigated and the diameters were determined and compared with the results of the angular measurements. The estimated diameter values in the angular and the spectral mode were in an excellent agreement with deviations of less than 0.20%.

Currently, photoplethysmograms (PPGs) are mostly used to determine a patient's blood oxygenation and pulse rate. However, PPG morphology conveys more information about the patient's cardiovascular status. Extracting this information requires measuring clean PPG waveforms that are free of artifacts. PPGs are highly susceptible to motion, which can distort the PPG-derived data. Part of the motion artifacts are considered to result from sensor-tissue motion and sensor deformation. It is hypothesized that these motion artifacts correlate with movement of the sensor with respect to the skin. This hypothesis has been proven true in a laboratory setup. In vitro PPGs have been measured in a skin perfusion phantom that is illuminated by a laser diode. Optical motion artifacts are generated in the PPG by translating the laser diode with respect to the PPG photodiode. The optical motion artifacts have been reduced significantly in vitro, by using a normalized least-mean-square algorithm with only a single coefficient that uses the laser's displacement as a reference for the motion artifacts. Laser displacement has been measured accurately via self-mixing interferometry by a compact laser diode with a ball lens integrated into the package, which can be easily integrated into a commercial sensor.

A novel balloon catheter-based diffusing optical device was designed and evaluated to assist in treating excessive menstrual bleeding. A synthetic fused-silica fiber was micro-machined precisely to create scattering segments on a 25 mm long fiber tip for uniform light distribution. A visible wavelength (λ = 532 nm) was used to specifically target the endometrium due to the high vascularity of the uterine wall. Optical simulation presented 30% wider distribution of photons along with approximately 40% higher irradiance induced by addition of a glass cap to the diffuser tip. Incorporation of the optical diffuser with a polyurethane balloon catheter considerably enhanced coagulation depth and area (i.e., 3.5 mm and 18.9 cm² at 1 min irradiation) in tissue in vitro. The prototype device demonstrated the coagulation necrosis of 2.8±1.2 mm (n=18) and no thermal damage to myometrium in in vivo caprine models. A prototype 5 cm long balloon catheter-assisted optical diffuser was also evaluated with a cadaveric human uterus to confirm the coagulative response of the uterine tissue as well as to identify the further design improvement and clinical applicability. The proposed catheter-based diffusing optical device can be a feasible therapeutic tool to photocoagulate endometrial cell layers in an efficient and safe manner.

Endotoxins released in the dental root by Gram-negative microorganisms can be neutralized by calcium hydroxide, when this medication is applied inside the root canal for at least seven days. However, several clinical situations demand faster root canal decontamination. Thus, for faster endotoxin neutralization, endodontists are seeking additional treatments. The in vitro study tested whether or not intracanal Nd:YAG laser irradiation would be able to neutralize endotoxin within the human dental root canal in a single session. Twenty-four human teeth with one root were mounted between two chambers. After conventional endodontic treatment, root canals were contaminated with Escherichia coli endotoxin. Then they were irradiated or not (controls) in contact mode with an Nd:YAG laser (1.5 W, 15 Hz, 100 mJ and pulse fluency of 124  J/cm²). The endotoxin activity was measured using the limulus lysate technique and data were statistically compared (p≤0.05). The concentration of active endotoxin measured in the negative control group was significantly lower than that of the positive control group (p=0.04). The concentrations of endotoxin in both irradiated groups were significantly lower than that of the positive control group (p=0.027) and similar to that of negative control group (p=0.20). A single session of intracanal Nd:YAG laser irradiation is able to neutralize endotoxin in the dental root tissues.

This article [J. Biomed. Opt.. 17, , 105009 (2012)] was originally published online on 18 October 2012 with an error in the author list. The name Albert Pinchasov was corrected to Albert Pinhasov, as it appears above. This article was corrected online on 5 November 2012.

This article [J. Biomed. Opt.. 17, , 117005 (2012)] was originally published online on 7 November 2012 with an error on p. 2.