Visualization of microvascular networks could provide new information about function and disease. We demonstrate the capabilities of a 30-MHz ultrasound array system for photoacoustic microscopy of small (≤300 μm) vessels in a rat. 3D images obtained by translating the array in the elevation direction are compared with photographs of excised skin. The system is shown to have 100-μm lateral resolution, 25-μm axial resolution, and 3-mm imaging depth. To our knowledge this is the first report on photoacoustic microscopy of the microvasculature with a high-frequency array transducer. It is anticipated that the system can be used for studying and diagnosing a number of diseases including cancer, atherosclerosis, dermatological disorders, and peripheral microvascular complications in diabetes.

The optoacoustic technique is a noninvasive imaging method with high spatial resolution. It potentially can be used to monitor anatomical and physiological changes. Photodynamic therapy (PDT)-induced vascular damage is one of the important mechanisms of tumor destruction, and real-time monitoring of vascular changes can have therapeutic signicance. A unique optoacoustic system is developed for neovascular imaging during tumor phototherapy. In this system, a single-pulse laser beam is used as the light source for both PDT and for concurrently generating ultrasound signals for optoacoustic imaging. To demonstrate its feasibility, this system is used to observe vascular changes during PDT treatment of chicken chorioallantoic membrane (CAM) tumors. The photosensitizer used in this study is protoporphyrin IX (PpIX) and the laser wavelength is 532 nm. Neovascularization in tumor angiogenesis is visualized by a series of optoacoustic images at different stages of tumor growth. Damage of the vascular structures by PDT is imaged before, during, and after treatment. Rapid, real-time determination of the size of targeted tumor blood vessels is achieved, using the time difference of positive and negative ultrasound peaks during the PDT treatment. The vascular effects of different PDT doses are also studied. The experimental results show that a pulsed laser can be conveniently used to hybridize PDT treatment and optoacoustic imaging and that this integrated system is capable of quantitatively monitoring the structural change of blood vessels during PDT. This method could be potentially used to guide PDT and other phototherapies using vascular changes during treatment to optimize treatment protocols, by choosing appropriate types and doses of photosensitizers and doses of light.

It is known that singlet oxygen (¹O₂is the main factor mediating cytotoxicity in photodynamic therapy (PDT). The effectiveness of a PDT treatment is directly linked to the ¹O₂ produced in the target. Although the luminescence from ¹O₂ is suggested as an indicator for evaluating photodynamic therapy, the inherent disadvantages limit its potential for in vivo applications. We have previously reported that chemiluminescence can be used to detect¹O₂ production in PDT and have linked the signal to the cytotoxicity. We further our investigation for monitoring ¹O₂ production during PDT. The lifetime of 3,7-dihydro-6-{4-[2-(N′-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo {1,2-a} pyrazin-3-one-chemiluminescence (FCLA-CL) is evaluated, and the results show that it is much longer than that of direct luminescence of ¹O₂. A gated measurement algorithm is developed to fully utilize the longer lifetime for a clean measurement of the CL without the interference from the irradiation light. The results show that it is practically feasible to use the technique to monitor the ¹O₂. Compared to the direct ¹O₂ luminescence measurement, our new technique is sensitive and can be realized with a conventional optical detector with excellent signal-to-noise ratio. It thus provides a means for real-time in vivo monitoring of ¹O₂ production during PDT.

Light and heat distributions are measured in a rat glioma model used in photodynamic therapy. A fiber delivering 632-nm light is fixed in the brain of anesthetized BDIX rats. Fluence rates are measured using calibrated isotropic probes that are positioned stereotactically. Mathematical models are then used to derive tissue optical properties, enabling calculation of fluence rate distributions for general tumor and light application geometries. The fluence rates in tumor-free brains agree well with the models based on diffusion theory and Monte Carlo simulation. In both cases, the best fit is found for absorption and reduced scattering coefficients of 0.57 and 28 cm^-1, respectively. In brains with implanted BT₄C tumors, a discrepancy between diffusion and Monte Carlo–derived two-layer models is noted. Both models suggest that tumor tissue has higher absorption and less scattering than normal brain. Temperatures are measured by inserting thermocouples directly into tumor-free brains. A model based on diffusion theory and the bioheat equation is found to be in good agreement with the experimental data and predict a thermal penetration depth of 0.60 cm in normal rat brain. The predicted parameters can be used to estimate the fluences, fluence rates, and temperatures achieved during photodynamic therapy.

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I₃₄₅/I₅₀₀ ratio (<10), while for malignant specimens, the I₃₄₅/I₅₀₀ ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

Nonmelanoma skin cancer is the most common form of human cancer, often resulting in high morbidity. Low visual contrast of these tumors makes their delineation a challenging problem. Employing a linearly polarized monochromatic light source and a wide-field CCD camera, we have developed a technique for fluorescence polarization imaging of the nonmelanoma cancers stained using antibiotics from the tetracycline family. To determine the feasibility of the method, fluorescence polarization images of 86 thick, fresh cancer excisions were studied. We found that the level of endogenous fluorescence polarization was much lower than that of exogenous, and that the average values of fluorescence polarization of tetracycline derivatives were significantly higher in cancerous as compared to normal tissue. Out of 86 tumors [54 stained in demeclocycline (DMN) and 32 in tetracycline (TCN)], in 79 cases (51—DMN, 28—TCN) the location, size, and shape of the lesions were identified accurately. The results of this trial indicate that nonmelanoma skin tumors can be distinguished from healthy tissue based on the differences in exogenous fluorescence polarization of TCN and/or DMN. Therefore, the developed technique can provide an important new tool for image-guided cancer surgery.

Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifacts due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system is developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consists of a constant UV-A light source, digital color camera, and head-positioning device. The system for acquisition of a fluorescent facial image and the image analysis method is described. The imaging modality provides uniform light distribution and presents a discernible color fluorescent image. Valuable parameters of sebum excretion are obtained after image analysis. The imaging system, which provides a noncontact method, is proved to be a useful tool to evaluate the amount and pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the described method is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time.

Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve® enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.

The use of highly specific and highly sensitive immunofluorescent probes is a promising approach for biomedical imaging in living tissue. We focus on immunofluorescence with quantum dot bioconjugates for hepatoma detection in vivo. We synthesized specific immunofluorescent probes by linking quantum dots to AFP (alpha-fetoprotein) antibody for specific binding AFP—an important marker for hepatocellular carcinoma cell lines. In in vivo studies, the characteristic quantum dot (QD) fluorescent property is exhibited by the QDs-Anti-AFP probes in tumor and they demonstrate active tumor targeting and spectroscopic hepatoma imaging with an integrated fluorescence imaging system. We investigate the inhomogeneous distribution of the QDs-Anti-AFP probes in tumor by using a site-by-site measurement method to test their ability for distribution studies of cancer cells. These results demonstrate the practicality of QD bioconjugates as attractive fluorescent probes for biomedical detection.

We explore the potential of optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) in a new area—whole organ imaging. The techniques are implemented on an in-house prototype benchtop system with improved image quality and the capacity to image larger samples (up to 3 cm) than previous systems based on stereo microscopes. Imaging performance tests confirm high geometrical accuracy, accurate relative measurement of linear attenuation coefficients, and the ability to image features at the 50-µm level. Optical labeling of organ microvasculature was achieved using two stains deposited via natural in vivo circulatory processes: a passive absorbing ink-based stain and an active fluorescin FITC-lectin conjugate. The lectin protein binds to the endothelial lining, and FITC fluorescense enables optical-ECT imaging. Three-dimensional (3-D) optical-CT images have been acquired of a normal rat heart and left lung and a mouse right lung showing exquisite detail of the functional vasculature and relative perfusion distribution. Coregistered optical-ECT images were also acquired of the mouse lung and kidney. Histological sections confirmed effective labeling of microvasculature throughout the organs. The advantages of optical-CT and optical-ECT include the potential for a unique combination of high resolution and high contrast and compatibility with a wide variety of optical probes, including gene expression labeling fluorescent reporter proteins.

Angularly resolved light scattering and wavelength-resolved darkfield scattering spectroscopy measurements were performed on intact, control EMT6 cells and cells stained with high-extinction lysosomal- or mitochondrial-localizing dyes. In the presence of the lysosomal-localizing dye NPe6, we observe changes in the details of light scattering from stained and unstained cells, which have both wavelength- and angular-dependent features. Analysis of measurements performed at several wavelengths reveals a reduced scattering cross section near the absorption maximum of the lysosomal-localizing dye. When identical measurements are made with cells loaded with a similar mitochondrial-localizing dye, HPPH, we find no evidence that staining mitochondria had any effect on the light scattering. Changes in the scattering properties of candidate populations of organelles induced by the addition of an absorber are modeled with Mie theory, and we find that any absorber-induced scattering response is very sensitive to the inherent refractive index of the organelle population. Our measurements and modeling are consistent with EMT6-cell-mitochondria having refractive indices close to those reported in the literature for organelles, approximately 1.4. The reduction in scattering cross section induced by NPe6 constrains the refractive index of lysosomes to be significantly higher. We estimate the refractive index of lysosomes in EMT6 cells to be approximately 1.6.

Confocal spectroscopic techniques are applied to selected Raman bands to study the microscopic features of acetabular cups made of ultra-high molecular weight polyethylene (UHMWPE) before and after implantation in vivo. The micrometric lateral resolution of a laser beam focused on the polymeric surface (or subsurface) enables a highly resolved visualization of 2-D conformational population patterns, including crystalline, amorphous, orthorhombic phase fractions, and oxidation index. An optimized confocal probe configuration, aided by a computational deconvolution of the optical probe, allows minimization of the probe size along the in-depth direction and a nondestructive evaluation of microstructural properties along the material subsurface. Computational deconvolution is also attempted, based on an experimental assessment of the probe response function of the polyethylene Raman spectrum, according to a defocusing technique. A statistical set of high-resolution microstructural data are collected on a fully 3-D level on γray irradiated UHMWPE acetabular cups both as-received from the maker and after retrieval from a human body. Microstructural properties reveal significant gradients along the immediate material subsurface and distinct differences are found due to the loading history in vivo, which cannot be revealed by conventional optical spectroscopy. The applicability of the confocal spectroscopic technique is valid beyond the particular retrieval cases examined in this study, and can be easily extended to evaluate in-vitro tested components or to quality control of new polyethylene brands. Confocal Raman spectroscopy may also contribute to rationalize the complex effects of γ-ray irradiation on the surface of medical grade UHMWPE for total joint replacement and, ultimately, to predict their actual lifetime in vivo.

Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.

Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10−18 L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.

Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer's disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.

Rapid near-infrared (NIR) diffuse optical tomography is implemented using a low-coherence source. The spectral bandwidth of the low-coherence source is dispersed and coupled to linearly bundled fibers, such that "spread"-spectral encoding among the bundled fibers is formed, and could be used for parallel source illumination onto tissue. In comparison with a previous spectral-encoding technique that employed multiple laser diodes, the use of a low-coherence source for spread-spectral encoding presents a few unique characteristics: (1) it provides shift-free spectral encoding; (2) it reduces the reconstruction uncertainty significantly owing to the minimization of spontaneous channel-to-channel intensity fluctuation; and (3) it enables the implementation of NIR tomography into an endoscopic imaging mode. A 20-mW superluminescent diode centered at 840 nm with a 40-nm bandwidth is used as the source, and a sampling speed of 5 Hz is obtained in a 27-mm imaging array consisting of eight sources and eight detection channels. The principles of using a low-coherence source for spread-spectral encoding are elaborated, the characteristic performances are demonstrated, and the preliminary results of imaging hemoglobin absorption variations during 10 s of voluntary breath-holding are presented.

Optical coherence tomography (OCT) is a powerful, noninvasive biomedical technique that uses low-coherence light sources to obtain in-depth scans of biological tissues. We report results obtained with three different sources emitting at 1570, 1330, and 810 nm, respectively. Attenuation and backscattering measurements are obtained with these sources for several in vitro biological tissues. From these measurements, we use a graphical method to make comparisons of the penetration depth and backscattering intensity of each wavelength for the studied samples. The influence of the coherence length of each source is also taken into account in order to make a more relevant comparison.

Optical Doppler tomography is a valuable functional extension of optical coherence tomography (OCT) that can be used to study subsurface blood flows of biological tissues. We propose a novel frequency estimation technique that uses an adaptive notch filter (ANF) to track the depth-resolved Doppler frequency. This new technique is a minimal-parameter filter and works in the time domain without the need of Fourier transformation. Therefore, the algorithm has a computationally efficient structure that may be well suited for implementation in real-time ODT systems. Our simulations and imaging results also demonstrate that this filter has good performance in terms of noise robustness and estimation accuracy compared with existing estimation algorithms.

We develop a dual-channel optical coherence tomography/indocyanine green (OCT/ICG) fluorescence system based on our previously reported ophthalmic OCT/confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of the ICG dye and light from the same optical source is used to generate the OCT image and to excite the ICG fluorescence. The system enables the clinician to visualize simultaneously en face OCT slices and corresponding ICG angiograms of the ocular fundus, displayed side by side. C-scan (constant depth) and B-scan (cross section) images are collected by fast en face scanning (T-scan). The pixel-to-pixel correspondence between the OCT and angiography images enables the user to precisely capture OCT B-scans at selected points on the ICG confocal images.

Light-activated resin-based dental composites are increasingly replacing dental amalgam. However, these materials are limited by inefficient setting reactions as a function of depth that constrain the maximum extent of cure. Insufficient curing can contribute to an overall reduction in biocompatibility of the material. We demonstrate dynamic refractive index measurements of a commercial dental composite throughout cure using spectral domain low coherence interferometry. Our results show a linear relationship between the change in refractive index and polymerization-induced reduction in physical thickness during light-activated curing. This relationship between the optical and physical density demonstrates the potential of this technique as a unique noninvasive tool for measurement of the conversion degree of curing dental composite materials.

The absorption spectrum of collagen powder is measured between 610 and 1040 nm by time-resolved transmittance spectroscopy. Absorption spectra of breast from healthy volunteers are then interpreted, adding collagen to the other absorbers previously considered (i.e., oxy- and deoxyhemoglobin, water, and lipids). A significant amount of collagen, depending on breast type, is estimated to be present. Adding collagen to the fitting procedure affects remarkably the estimated values of blood content and oxygenation. The quantification of collagen has potential implications for the assessment of breast density and cancer risk.

The development of photodynamic therapy into a modality for treatment of prostate cancer calls for reliable optical dosimetry. We employ, for the first time, interstitial time-resolved spectroscopy to determine in vivo optical properties of human prostate tissue. Nine patients are included in the study, and measurements are conducted prior to primary brachytherapy treatment of prostate cancer. Intrasubject variability is examined by measuring across three tissue volumes within each prostate. The time-resolved instrumentation proves its usefulness by producing good signal levels in all measurements. We are able to present consistent values on reduced scattering coefficients (μ_s^′, absorption coefficients (μ_a), and effective attenuation (μ_eff) at the wavelengths 660, 786, and 916 nm. At 660 nm, μ_s^′ is found to be 9±2 cm^-1, and μa is 0.5±0.1 cm^-1. Derived values of μ_eff are in the range of 3 to 4 cm^-1 at 660 nm, a result in good agreement with previously published steady state data. Total hemoglobin concentration (THC) and oxygen saturation are spectroscopically determined using derived absorption coefficients. Derived THC values are fairly variable (215±65 μM), while derived values of oxygen saturation are gathered around 75% (76±4%). Intrasubject variations in derived parameters correlate (qualitatively) with the heterogeneity exhibited in acquired ultrasound images.

We study the behavior of optical properties of human skin across its layers. We describe ellipsometric measurements for spectrally resolved in vivo investigations of biological tissue. We show in vivo measurements on human skin of the ellipsometric parameters Ψ and Δ, which describe the change in polarization of light on reflection on a sample. A tape-stripping study reveals the depth profile of the ellipsometric parameters into the stratum corneum. The depth profile shows an increase in both quantities with increased depth. Analyzing the development of Ψ on the number of strips using an exponential function shows a steady state after approximately 2 μm. The evolution of Ψ and Δ can be described using a morphological model containing an effective medium approximation accounting for the water content of the skin, surface roughnesses of the corneocytes, as well as the alternating cell and lipid layers.

The optical parameters absorption coefficient, scattering coefficient, and the anisotropy factor of platelets (PLTs) suspended in plasma and cell-free blood plasma are determined by measuring the diffuse reflectance, total and diffuse transmission, and subsequently by inverse Monte Carlo simulation. Furthermore, the optical behavior of PLTs and red blood cells suspended in plasma are compared with those suspended in saline solution. Cell-free plasma shows a higher scattering coefficient and anisotropy factor than expected for Rayleigh scattering by plasma proteins. The scattering coefficient of PLTs increases linearly with the PLT concentration. The existence of physiological concentrations of leukocytes has no measurable influence on the absorption and scattering properties of whole blood. In summary, red blood cells predominate over the other blood components by two to three orders of magnitude with regard to absorption and effective scattering. However, substituting saline solution for plasma leads to a significant increase in the effective scattering coefficient and therefore should be taken into consideration.

^{Photodynamic therapy (PDT) is an alternative to radical surgical resection for T1a or nonresectable carcinomas of the gastroesophageal junction. Besides the concentration of the photosensitizer, the light distribution in tissue is responsible for tumor destruction. For this reason, knowledge about the behavior of light in healthy and dysplastic tissue is of great interest for careful irradiation scheduling. The aim of this study is to determine the optical parameters (OP) of healthy and carcinomatous tissue of the gastroesophageal junction in vitro to provide reproducible parameters for optimal dosimetry when applying PDT. A total of 36 tissue samples [adenocarcinoma tissue (n=21), squamous cell tissue (n=15)] are obtained from patients with carcinomas of the gastroesophageal junction. The optical parameters are measured in 10-nm steps using new integrating sphere spectrometers in the PDT-relevant wavelength range of 300 to 1140 nm and evaluated by inverse Monte-Carlo simulation. Additional examinations are done in healthy tissue from the surgical safety margin. In the wavelength range of frequently applied photosensitizers at 330, 630, and 650 nm, the absorption coefficient in tumor tissue (adenocarcinoma 1.22, 0.16, and 0.15 mm-1; squamous cell carcinoma 1.48, 0.13, and 0.11 mm-1) is significantly lower than in healthy tissue (stomach 3.34, 0.26, and 0.20 mm-1; esophagus 2.47, 0.21, and 0.18 mm-1). The scattering coefficient of all tissues decreases continuously with increasing wavelength (adenocarcinoma 22.8, 12.99, and 12.52 mm-1; squamous cell carcinoma 19.44, 9.35, and 8.98 mm-1; stomach 20.55, 13.96, and 13.94 mm-1; esophagus 20.34, 12.56, and 12.22 mm-1. All tissues show an anisotropy factor between 0.80 and 0.94 over the entire spectrum. (Abstract truncated).}

We investigate anisotropic light propagation in biological tissue in steady-state and time domains. Monte Carlo simulations performed for tissue that consists of aligned cylindrical and spherical scatterers show that steady-state and time-resolved reflectance depends strongly on the measurement direction relative to the alignment of the cylinder axis. We examine the determination of optical properties using an isotropic diffusion model and find that in the time domain, in contrast to steady-state spatially resolved reflectance measurements, the obtained absorption coefficient does not depend on the measurement direction and is close to the true value. Contrarily, the derived reduced scattering coefficient depends strongly on the measurement direction in both domains. Measurements of the steady-state and time-resolved reflectance from bovine tendon confirm the theoretical findings.

Recently, the field of photoacoustic tomography has experienced considerable growth. Although several commercially available pure optical imaging modalities, including confocal microscopy, two-photon microscopy, and optical coherence tomography, have been highly successful, none of these technologies can penetrate beyond ~1 mm into scattering biological tissues because all of them are based on ballistic and quasiballistic photons. Consequently, heretofore there has been a void in high-resolution optical imaging beyond this depth limit. Photoacoustic tomography has filled this void by combining high ultrasonic resolution and strong optical contrast in a single modality. However, it has been assumed in reconstruction of photoacoustic tomography until now that ultrasound propagates in a boundary-free infinite medium. We present the boundary conditions that must be considered in certain imaging configurations; the associated inverse solutions for image reconstruction are provided and validated by numerical simulation and experiment. Partial planar, cylindrical, and spherical detection configurations with a planar boundary are covered, where the boundary can be either hard or soft. Analogously to the method of images of sources, which is commonly used in forward problems, the ultrasonic detectors are imaged about the boundary to satisfy the boundary condition in the inverse problem.

The spectral analysis and classification for discrimination of pulsed laser-induced autofluorescence spectra of pathologically certified normal, premalignant, and malignant oral tissues recorded at a 325-nm excitation are carried out using MATLAB@R6-based principal component analysis (PCA) and k-means nearest neighbor (k-NN) analysis separately on the same set of spectral data. Six features such as mean, median, maximum intensity, energy, spectral residuals, and standard deviation are extracted from each spectrum of the 60 training samples (spectra) belonging to the normal, premalignant, and malignant groups and they are used to perform PCA on the reference database. Standard calibration models of normal, premalignant, and malignant samples are made using cluster analysis. We show that a feature vector of length 6 could be reduced to three components using the PCA technique. After performing PCA on the feature space, the first three principal component (PC) scores, which contain all the diagnostic information, are retained and the remaining scores containing only noise are discarded. The new feature space is thus constructed using three PC scores only and is used as input database for the k-NN classification. Using this transformed feature space, the centroids for normal, premalignant, and malignant samples are computed and the efficient classification for different classes of oral samples is achieved. A performance evaluation of k-NN classification results is made by calculating the statistical parameters specificity, sensitivity, and accuracy and they are found to be 100, 94.5, and 96.17%, respectively.

A Monte Carlo model for polarized light propagation in birefringent, optically active, multiply scattering media is developed in an effort to accurately represent the propagation of polarized light in biological tissue. The model employs the Jones N-matrix formalism to combine both linear birefringence and optical activity into a single effect that can be applied to photons as they propagate between scattering events. Polyacrylamide phantoms with strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres as scattering particles are used for experimental validation. Measurements are made using a Stokes polarimeter that detects scattered light in different geometries, and compared to the results of Monte Carlo simulations run with similar parameters. The results show close agreement between the experimental measurements and Monte Carlo calculations for phantoms exhibiting turbidity and birefringence, as well as for phantoms exhibiting turbidity, birefringence, and optical activity. Other scattering-independent polarization properties can be incorporated into the developed Jones N-matrix formalism, enabling quantification of the polarization effects via an accurate polarization-sensitive Monte Carlo model.

The use of perturbation and differential Monte Carlo (pMC/dMC) methods in conjunction with nonlinear optimization algorithms were proposed recently as a means to solve inverse photon migration problems in regionwise heterogeneous turbid media. We demonstrate the application of pMC/dMC methods for the recovery of optical properties in a two-layer extended epithelial tissue model from experimental measurements of spatially resolved diffuse reflectance. The results demonstrate that pMC/dMC methods provide a rapid and accurate approach to solve two-region inverse photon migration problems in the transport regime, that is, on spatial scales smaller than a transport mean free path and in media where optical scattering need not dominate absorption. The pMC/dMC approach is found to be effective over a broad range of absorption (50 to 400%) and scattering (70 to 130%) perturbations. The recovery of optical properties from spatially resolved diffuse reflectance measurements is examined for different sets of source-detector separation. These results provide some guidance for the design of compact fiber-based probes to determine and isolate optical properties from both epithelial and stromal layers of superficial tissues.

Corneal laser welding is a technique used clinically to induce the immediate sealing of corneal wounds. We present an experimental and model analysis of the temperature dynamics during diode laser-induced corneal welding, which is aimed at characterizing the mechanism of tissue fusion. Ex vivo tests were performed on porcine eyes in the typical irradiation conditions used for laser-induced suturing in cornea transplant. Three laser power densities (12.5 W/cm², 16.7 W/cm², 20.8 W/cm²) were tested. The superficial temperature of the cornea was measured by means of an infrared thermocamera. Experimental data were compared with the results of a three-dimensional (3D) model of a laser-welding process in the cornea, solved by the use of the Finite Element Method (FEM). The model solution and experimental results showed good agreement. The model was thus used to estimate the temperature enhancement inside the corneal wound and to calculate the thermal damage inside the tissue. The results indicated a selective, spatially confined heating effect that occurred at operative temperatures (59 to 66°C) close to intermediate denaturation points of the stromal collagen, before its complete disorganization. No significant heat damage to the region of the laser-treated wound was evidenced in the operative irradiation conditions of corneal welding.

We present a physical model to describe iontophoresis time recordings. The model is a combination of monodimensional material diffusion and decay, probably due to transport by blood flow. It has four adjustable parameters, the diffusion coefficient, the decay constant, the height of the response, and the shot saturation constant, a parameter representing the relative importance of subsequent shots (in case of saturation). We test the model with measurements of blood perfusion in the capillary bed of the fingers of women who recently had preeclampsia and in women with a history of normal pregnancy. From the fits to the measurements, we conclude that the model provides a useful physical description of the iontophoresis process.

We describe a near-infrared spectroscopy (NIRS) method to noninvasively measure relative changes in the pulsate components of cerebral blood flow (pCBF) and volume (pCBV) from the shape of heartbeat oscillations. We present a model that is used and data to show the feasibility of the method. We use a continuous-wave NIRS system to measure the arterial oscillations originating in the brains of piglets. Changes in the animals' CBF are induced by adding CO₂ to the breathing gas. To study the influence of scalp on our measurements, comparative, invasive measurements are performed on one side of the head simultaneously with noninvasive measurements on the other side. We also did comparative measurements of CBF using a laser Doppler system to validate the results of our method. The results indicate that for sufficient source-detector separation, the signal contribution of the scalp is minimal and the measurements are representative of the cerebral hemodynamics. Moreover, good correlation between the results of the laser Doppler system and the NIRS system indicate that the presented method is capable of measuring relative changes in CBF. Preliminary results show the potential of this NIRS method to measure pCBF and pCBV relative changes in neonatal pigs.

The investigation of flow profiles in microstructures and tissues by fluorescence correlation spectroscopy (FCS) has been a challenging topic in the past decade. Due to its inherent optical configuration, a circular focused laser beam, FCS is unable to resolve microfluidic flow directions. Earlier schemes reported the use of two laser beams or the use of nonsymmetrical laser foci to break the symmetry of the measurement system. This, however, is difficult to combine with confocal systems since it would require modifications that interfere with the imaging capabilities. We propose a method called line-scan FCS to measure different flow angles in microchannels and tissues. This method is implemented on a combined laser scanning confocal microscopy (LSCM) and FCS system that enables uncompromised imaging and spectroscopy measurements. We demonstrate that by scanning the laser beam with a defined speed and direction we can measure flow direction with the current system at an optimal resolution of at least 3 µm. The combination system is assessed by measuring flow profiles in a microchannel with and without obstruction. To extend the technique to live tissue measurements we demonstrate that line-scan FCS can determine the flow direction in zebrafish small blood vessels in a label-free approach.

Optical imaging using voltage-sensitive dyes has become an important tool for studying vortex-like electrical waves in the heart. Such waves, known as spiral or scroll waves, can spontaneously form in pathological ventricular myocardium, causing ventricular fibrillation and sudden death. Until recently, observations of scroll waves were limited to their surface manifestations, thus providing little information about the shape and location of their organizing center, the filament. We use computer modeling to assess the feasibility of visualizing filaments using dynamic transillumination imaging in conjunction with near-IR voltage-sensitive absorptive dyes (absorptive transillumination). We simulate transillumination signals produced by the intramural scroll waves in a realistic slab of ventricular tissue with trabeculated endocardial surface. The computations use a detailed ionic model of electrical excitation (LRd) coupled to a photon transport model for cardiac tissue. Our simulations show that dynamic absorptive transillumination data, with subsequent processing involving either amplitude maps, time-space plots, or power-of-the-dominant-frequency maps, can be used to reliably detect intramural scroll waves through the whole thickness (~10 mm) of the ventricular wall. Neither variations in the thickness of the myocardial wall nor noise impeded the detection of intramural filaments.

Retinal imaging spectroscopy can provide functional maps using chromophore spectra. For example, oxygen saturation maps show ischemic areas from diabetes and venous occlusions. Obtaining retinal spatial-spectral data has been difficult due to saccades and long data acquisition times (>5 s). We present a snapshot imaging spectrometer with far-reaching applicability that acquires a complete spatial-spectral image cube in ~3 ms from 450 to 700 nm with 50 bands, eliminating motion artifacts and pixel misregistration. Current retinal spectral imaging approaches are incapable of true snapshot operation over a wide spectral range with a large number of spectral bands. Coupled to a fundus camera, the instrument returns true color retinal images for comparison to standard fundus images and for image validation while the patient is still dilated. Oxygen saturation maps were obtained with a three-wavelength algorithm: for healthy subjects arteries were ~95% and veins 30 to 35% less. The instrument is now undergoing clinical trials.

Off-the-shelf spatial light modulators (SLMs) like those commonly included in video projection devices have been seldom used for the compensation of eye aberrations, mainly due to the relatively low dynamic range of the phase retardation that can be introduced at each pixel. They present, however, some interesting features, such as high spatial resolution, easy handling, wide availability, and low cost. We describe an efficient four-level phase encoding scheme that allows us to use conventional SLMs for compensating optical aberrations as those typically found in human eyes. Experimental results are obtained with artificial eyes aberrated by refractive phase plates introducing either single Zernike terms or complex eye aberration patterns. This proof-of-concept is a step toward the use of low-cost, general purpose SLMs for the compensation of eye aberrations.

A highly parallel microarray scanner for functional genomic research using a dynamically reconfigurable holographic excitation and hyperspectral fluorescence detection is described. The light from two laser sources (405 and 532 nm) is split into an arbitrary number of focused spots on the biochip using a pair of spatial light modulators and a novel imaging system. The parallel optical scanner includes a hyperspectral detection unit with a high-sensitivity CCD camera to detect and analyze the emitted fluorescence spectra (from 430 to 800 nm) of all illuminated spots simultaneously. By using an xy scanner, the spectra of all spots on an entire array can be read out line by line.

Several well-established techniques are available to obtain 3-D image information of biomedical specimens, each with their specific advantages and limitations. Orthogonal plane fluorescence optical sectioning (OPFOS), or selective plane illumination microscopy (SPIM), are additional techniques which, after adequate specimen preparation, produce high quality, autoaligned sectional images in nearly real time, of bone as well as soft tissue. Up until now, slicing resolutions down to 14 μm have been obtained. We present a high resolution (HR) OPFOS method, which delivers images that approach the quality of histological sections. With our HROPFOS technique, we achieve in-plane resolutions of 1 μm and a slicing resolution of 2 μm. A region of interest within an intact and much larger object can be imaged without problems, and as the optical technique is nondestructive, the object can be measured in any slicing direction. We present quantitative measurements of resolution. A 3-D model reconstructed from our HROPFOS data is compared to SEM results, and the technique is demonstrated with section images and 3-D reconstructions of middle ear specimens.

A dual-wavelength microarray scanner based on laser confocal principle is constructed to acquire the laser-induced two-color fluorescence from dyes Cy3 and Cy5. Unlike most commercially available micoarray scanners whose lasers are continuous wave, filters are bandpass and the channels are multiple photomultiplier tubes (PMTs), the system mainly consists of two diode lasers, four volume holographic band-stop filters, and one PMT. The central wavelengths of two kinds of volume holographic band-stop filters are equal to those of the two laser, respectively. The fluorescence beams can pass through the filters, while laser beams are blocked by them because the filters are cut off. The two fluorescences can pass through a common optical path. Thus, only one pinhole and one PMT are required. The two fluorescences are acquired by the PMT with time sharing. The two respective fluorescences can be gathered when two diode lasers work asynchronously. If lasers work synchronously, the two wavelength fluorescences could be acquired simultaneously by the same PMT. A synthesis of a two-color fluorescence image can be realized straightforwardly in this manner. An experiment is conducted and Cy3 and Cy5 fluorescences are captured. Gridding of the fluorescent images based on basic morphological erosion appropriately locates the boundary between adjacent spots.