Selective tissue damage on the cellular level can be achieved by microbubble formation around laser-heated intracellular pigments. To acquire a more detailed understanding of the laser tissue interaction in the highly pigmented retinal pigment epithelium (RPE), we irradiate aqueous suspensions of absorbing microparticles by short pulsed laser irradiation (12 ns, 240 ns, and 1.8 µs). Porcine retinal pigment epithelial melanosomes, gold beads, and magnetic silica beads are used as absorbers. Pulsed laser heating of the particles leads to vaporization of the surrounding liquid. The resulting transient microbubbles on the particle surface are imaged directly on a microscopic level by fast flash light photography. Furthermore, the bubble dynamics is probed by a low power laser. Threshold radiant exposures for bubble formation and nucleation temperatures are experimentally determined. Superheating of the surrounding water to 150 °C for melanosomes and to more than 200 °C for magnetic silica beads (=3 µm) and gold beads (=500 nm) is achieved. With these data, the absorption coefficient of a melanosome is calculated by thermal modeling of the experimental thresholds for bubble formation.

In quantitative evaluation of facial skin chromophore content using color imaging, several factors such as view angle and facial curvature affect the accuracy of measured values. To determine the influence of view angle and facial curvature on the accuracy of quantitative image analysis, we acquire cross-polarized diffuse reflectance color images of a white-patched mannequin head model and human subjects while varying the angular position of the head with respect to the image acquisition system. With the mannequin head model, the coefficient of variance (CV) is determined to specify an optimal view angle resulting in a relatively uniform light distribution on the region of interest (ROI). Our results indicate that view angle and facial curvature influence the accuracy of the recorded color information and quantitative image analysis. Moreover, there exists an optimal view angle that minimizes the artifacts in color determination resulting from facial curvature. In a specific ROI, the CV is less in smaller regions than in larger regions, and in relatively flat regions. In clinical application, our results suggest that view angle affects the quantitative assessment of port wine stain (PWS) skin erythema, emphasizing the importance of using the optimal view angle to minimize artifacts caused by nonuniform light distribution on the ROI. From these results, we propose that optimal view angles can be identified using the mannequin head model to image specific regions of interest on the face of human subjects.

Our purpose is to correct digital CCD-recorded Scheimpflug photographs, imaging both the anterior and posterior corneal surface, the anterior chamber, and the anterior eye lens surface for optical distortions. In a ray-tracing algorithm the imaging of the posterior corneal surface in a given Scheimpflug photograph is corrected by applying Snell's law on parallel incident rays entering through the anterior corneal surface. Once the posterior corneal surface is corrected, the procedure is repeated, again with parallel incident rays entering through both the anterior and now corrected posterior corneal surface, to correct the imaging of the anterior eye lens surface. The refractive indices necessary for Snell's law are taken from Gullstrand's exact schematic eye model. Due to the optical/refractive correction, the digital Scheimpflug photograph decreases in size perpendicular to the direction of the optical axis. As a consequence the curvature radii of both the posterior corneal surface and the anterior lens surface are reduced significantly, as compared to the original digital Scheimpflug photograph. Furthermore, the corneal thickness and the anterior chamber depth are increased. The presented refractive correction method enables us to extract from Scheimpflug photographs the following quantities rather realistically: structure coordinates and curvature radii of both the posterior corneal surface and the anterior lens surface, corneal thickness, and anterior chamber depth. This method can readily be applied to other imaged quantities, such as the posterior eye lens surface, the lens thickness, and the pupillary opening.

An experimental study evaluating the application of laser welding of the cornea and the subsequent healing process is presented. The welding of corneal wounds is achieved after staining the cut walls with a solution of the chromophore indocyanine green, and irradiating them with a diode laser (810 nm) operating at low power (60 to 90 mW). The result is a localized heating of the cut, inducing controlled welding of the stromal collagen. In order to optimize this technique and to study the healing process, experimental tests, simulating cataract surgery and penetrating keratoplasty, were performed on rabbits: conventional and laser-induced suturing of corneal wounds were thus compared. A follow-up study 7 to 90 days after surgery was carried out by means of objective and histological examinations, in order to optimize the welding technique and to investigate the subsequent healing process. The analyses of the laser-welded corneas evidenced a faster and more effective restoration of the architecture of the stroma. No thermal damage of the welded stroma was detected, nor were there foreign body reactions or other inflammatory processes.

Optical molecular imaging could potentially enable noninvasive high-resolution characterization and diagnosis of living tissue. The capability to image multiple molecular targets simultaneously is particularly important. Currently this task cannot be achieved using conventional optical contrast agents, due to their broad spectral responses (~80 to 200 nm). Developments in research on semiconductor nanocrystals (quantum dots) provide one possible solution. We describe a different concept of multilabel molecular imaging that utilizes resonant light-scattering spectroscopy of multilayered nanospheres to achieve tunable ultrasharp resonance peaks with widths as narrow as 10 nm. Our theoretical study demonstrates that dozens of molecular targets can potentially be imaged simultaneously using this approach.

Photobiological research in the last 30 yr has shown the existence of ultraweak photon emission in biological tissue, which can be detected with sophisticated photomultiplier systems. Although the emission of this ultraweak radiation, often termed biophotons, is extremely low in mammalian cells, it can be efficiently increased by ultraviolet light. Most recently it was shown that UV-A (330 to 380 nm) releases such very weak cell radiation in differentiated human skin fibroblasts. Based on these findings, a new and powerful tool in the form of UV-A-laser-induced biophotonic emission of cultured cells was developed with the intention to detect biophysical changes between carcinogenic and normal cells. With suspension densities ranging from 1 to 8×106 cells/mL, it was evident that an increase of the UV-A-laser-light induced photon emission intensity could be observed in normal as well as melanoma cells. Using this new detection procedure of ultraweak light emission, photons in cell suspensions as low as 100 µL could be determined, which is a factor of 100 lower compared to previous procedures. Moreover, the detection procedure has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 µs instead of 150 ms, as reported in previous procedures. This improvement leads to measurements of light bursts up 107 photons/s instead of several hundred as found with classical designs. Overall, we find decreasing induction ratings between normal and melanoma cells as well as cancer-prone and melanoma cells. Therefore, it turns out that this highly sensitive and noninvasive device enables us to detect high levels of ultraweak photon emission following UV-A-laser-induced light stimulation within the cells, which enables future development of new biophysical strategies in cell research.

The effect of photodynamic therapy treatments depends on the concentration of photosensitizer at the treatment site; thus a simple method to quantify concentration is desirable. This study compares the concentration of a fluorophore and sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS4), measured by two simple fluorescence-based techniques in vivo to post mortem chemical extraction and fluorometric assay of those tissues: skin, muscle, fascia, liver, and kidney (cortex and medulla). Fluorescence was excited and detected by a single optical fiber, or by an instrument that measured the ratio of the fluorescence and excitation reflectance. The in vivo measurements were compared to calibration measurements made in tissue-simulating phantoms to estimate the tissue concentrations. Reasonable agreement was observed between the concentration estimates of the two instruments in the lighter colored tissues (skin, muscle, and fascia). The in vivo measurements also agreed with the chemical extractions at low (<0.6 µg/g) tissue concentrations, but underestimated higher tissue concentrations. Measurements of fluorescence lifetime in vivo demonstrated that AlPcS4 retains its mono-exponential decay in skin, muscle, and fascia tissues with a lifetime similar to that measured in aqueous tissue-simulating phantoms. In liver and kidney an additional short lifetime component was evident.

Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6-carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.

We develop an optical fluorescent mapping system that is able to record the action potential wavefront propagation within cardiac tissue samples with high spatial and temporal resolutions. The system's main component, the fluorescence acquisition device (customized CCD camera), offers a high spatial resolution of 128×128 pixels, with 12-bit digitization and a frame rate of 490 frames/s. The system is designed and implemented to image an area of approximately 20×20 mm at its minimum object distance of 140 mm, corresponding to a spatial resolution of approximately 3 line pairs/mm. Experiments using this system with di-4-ANEPPS-stained canine cardiac tissues with stimulated action potentials through external electrodes result in successful mappings of the distribution and propagation of the action potential wavefronts, showing the system's sensitivity to the change in fluorescence intensity in regions of action potentials. These data demonstrate this optical mapping system as a powerful device in the study of cardiac arrhythmia mechanisms.

We develop a compact scanning head for use in laser confocal fluorescence microscopy for in situ fluorescence imaging of organs. The head, cylindrical in shape, has 3.5 mm diameter and 30 mm length, and is thus small enough to operate in a living rat heart. The lateral and axial resolutions, defined as full widths at half maximum (FWHM) of a point spread function (PSF), measures 1.0 and 5.0 µm, respectively, for 488-nm excitation and 1.0 and 5.4 µm, respectively, for 543-nm excitation. The chromatic aberration between 488- and 543-nm laser beams is well suppressed. We perform Ca2+ imaging in cardiomyocytes through the right ventricular chamber of a perfused rat heart in line-scan mode with 2.9-ms time resolution. We also carried out two-color imaging of a fixed mouse heart and liver with subcellular resolution. The compact head of the microscope equipped with a line-scan imaging mode and two-color imaging mode is useful for in situ imaging in living organs with subcellular resolution and can advantageously be applied to in vivo research.

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

The spatial distribution of neurodegeneration in brains is difficult to visualize when working from 2-D serial slices. In studies where repetitive operant behavior measurements are made over several weeks following organic solvent exposure, definitive evidence of degeneration in brain structures may have been significantly cleared by the time the tissue is prepared histologically. The only remaining evidence that injury has occurred may be nothing more than neuronal and cellular debris. By choosing stains that are specific for this type of residual and/or indicative of specific pathology, a 3-D representation of the spatial distribution of the neuronal and cellular debris fields within the organ can be highlighted and displayed. We present a method for visualizing the spatial distribution of neuronal degeneration that can result from low-level organic solvent exposure scenarios. A cupric-silver stain highly specific for neuronal degeneration is used to identify neuronal debris fields in 73 serial slices of brains of rodents that were exposed to toluene vapors. Serial brain sections stained with cupric-silver are scanned at 600 dpi using a gray-scale protocol. Using commercially available software, scans are assembled into 3-D images showing both topographical and internal anatomical details. The reassembled images are further processed into stereo pairs. Gray-scale scans are compared to the original sections to establish gray-scale ranges for healthy and damaged tissue and artifact staining.

The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.

Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. By detecting nonlinear phenomena such as SHG and TPEF it is feasible to extract valuable information concerning the structure and the function of nematode neurons.

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.

Confocal microscopy (CM) is a powerful image acquisition technique that is well established in many biological applications. It provides 3-D acquisition with high spatial resolution and can acquire several different channels of complementary image information. Due to the specimen extraction and preparation process, however, the shapes of imaged objects may differ considerably from their in vivo appearance. Magnetic resonance microscopy (MRM) is an evolving variant of magnetic resonance imaging, which achieves microscopic resolutions using a high magnetic field and strong magnetic gradients. Compared to CM imaging, MRM allows for in situ imaging and is virtually free of geometrical distortions. We propose to combine the advantages of both methods by unwarping CM images using a MRM reference image. Our method incorporates a sequence of image processing operators applied to the MRM image, followed by a two-stage intensity-based registration to compute a nonrigid coordinate transformation between the CM images and the MRM image. We present results obtained using CM images from the brains of 20 honey bees and a MRM image of an in situ bee brain.

Temporal and spatial orchestration of neurovascular coupling in brain neuronal activity is crucial for comprehending the mechanism of functional cerebral metabolism and pathophysiology. Laser speckle contrast imaging (LSCI) through a thinned skull over the somatosensory cortex is utilized to map the spatiotemporal characteristics of local cerebral blood flow (CBF) in anesthetized rats during sciatic nerve stimulation. The time course of signals from all spatial loci among the massive dataset is hard to analyze, especially for the thousands of images, each of which composes millions of pixels. We introduce a temporal clustering analysis (TCA) method, which is proven as an efficient method to analyze functional magnetic resonance imaging (fMRI) data in the temporal domain. The timing and location of CBF activation shows that contralateral hindlimb sensory cortical microflow is activated to increase promptly in less than 1 s after the onset of 2-s electrical stimulation and is evolved in different discrete regions. This pattern is similar but slightly elaborated from the results obtained from laser Doppler flowmetry (LDF) and fMRI. We present this combination to investigate interacting brain regions, which might lead to a better understanding of the nature of brain parcellation and effective connectivity.

Mineralized biological materials have complex hierarchical graded structures. It is therefore difficult to understand the relations between their structure and mechanical properties. We report the use of electronic speckle pattern-correlation interferometry (ESPI) combined with a mechanical compression apparatus to measure the strain and Young's modulus of root dentin compressed under water. We describe the optomechanical instrumentation, experimental techniques and procedures needed to measure cubes as small as 1×1×2 mm. Calibration of the method is performed using aluminum, which shows that the measurements are accurate within 3% of the compression modulus reported for standard aluminum 6061. Our results reveal that the compression moduli of root dentin from the buccal and lingual sides of the root are quite different from the moduli of the interproximal sides. Root dentin from interproximal locations is found to have an average modulus of 21.3 GPa, which is about 40% stiffer than root dentin from the buccal and lingual locations, found to have a modulus of 15.0 GPa. Our approach can be used to map deformations on irregular surfaces, and measure strain on wet samples of varying sizes. This can be extended to the study of other biological materials including bone and synthetic biomaterials.

Numerical simulations of light scattering by a biconcave shaped human red blood cell (RBC) are carried out using the finite-difference time-domain (FDTD) method. A previously developed FDTD code for the study of light scattering by ice crystals is modified for the current purpose and it is validated against Mie theory using a spherically shaped RBC. Numerical results for the angular distributions of the Mueller scattering matrix elements of an RBC and their dependence on shape, orientation, and wavelength are presented. Also calculated are the scattering and absorption efficiencies. The implication of these results on the possibility of probing RBC shape changes is discussed.

Quantitative analysis of blood oxygen saturation using near-IR spectroscopy is made difficult by uncertainties in both the absolute value and the wavelength dependence of the optical path length. We introduce a novel means of assessing the wavelength dependence of path length, exploiting the relative intensities of several absorptions exhibited by an exogenous contrast agent (neodymium). Combined with a previously described method that exploits endogenous water absorptions, the described technique estimates the absolute path length at several wavelengths throughout the visible/near-IR range of interest. Isolated rat hearts (n=11) are perfused separately with Krebs-Henseleit buffer (KHB) and a KHB solution to which neodymium had been added, and visible/near-IR spectra are acquired using an optical probe made up of emission and collection fibers in concentric rings of diameters 1 and 3 mm, respectively. Relative optical path lengths at 520, 580, 679, 740, 800, 870, and 975 nm are 0.41±0.13, 0.49±0.21, 0.90±0.09, 0.94±0.01, 1.00, 0.84±0.01, and 0.78±0.08, respectively. The absolute path length at 975 nm is estimated to be 3.8±0.6 mm, based on the intensity of the water absorptions and the known tissue water concentration. These results are strictly valid only for the experimental geometry applied here.

Characterization of human tissue using near-IR (NIR) light is becoming increasingly popular. The light signal transmitted from the tissue contains information concerning inhomogeneities in tissue, such as size, position, and pathological states (benign or malignant). We discuss the most probable diffuse path (MPDP) related to frequency-domain diffuse photon density waves (DPDWs) propagating inside turbid media. We find that for a medium of finite size, the existence of boundaries between tissue and nonscattering media would have considerable impact on the path shape. It is also demonstrated that such paths can be used to obtain higher accuracy in localizing absorbers embedded in a homogeneous background. Based on the proposed MPDP, a new method for 3-D localization of heterogeneities in turbid media is proposed, which is validated by experiments using Intralipid and pork fat. The experiments are performed with an NIR breast cancer detection system designed and assembled in our lab, using 780-nm NIR light. In Intralipid, when the size of a single absorber is less than 1 cm, the localization error is about 2 mm. The results from pork fat are also acceptable.

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F color-center pulsed laser. A minimum absorbance detection level of 2.57×10–4 is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23±7.58×106 M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631±0.432×105 M. Stoichiometric binding coefficients of 1.530±0.981 for layer one and 1.792±0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important.

Near-infrared spectroscopy (NIRS) has been used for measurement of cerebral hemoglobin (Hb) concentrations in neonates to study cerebral oxygenation and hemodynamics. We perform measurements by portable three-wavelength NIR time-resolved spectroscopy (TRS) in a piglet hypoxia model with various degrees of oxygenation to estimate the absorption coefficient (µa) and reduced scattering coefficient (µ) of the head. Measurements of absolute values of µa at three wavelengths enable estimation of Hb concentration and Hb oxygen saturation in the head (SO2). However, there is a problem concerning which background absorption should be used to estimate Hb concentration in the head derived from µa at three wavelengths because it is different from a simple in vitro model. Therefore, we use two different background absorption values with the assumption that background absorption is due only to 85% (by volume) water or that background absorption is equal to absorption of the piglet head with blood exchange transfusion by fluorocarbon (FC), and we compared SO2 measured by TRS with arterial Hb oxygen saturation (SaO2) and sagittal sinus venous Hb oxygen saturation (SvO2) measured by a co-oximeter at several inspired fractional O2(FIO2) concentrations. We find that SO2 values using the absorption (abs) of the piglet head with blood exchange transfusion (BET) by FC are not significantly different from SO2 values using the water-only background at FIO2 in the range of 15 to 100%, but that the values using abs of the head with BET by FC are lower than the values using the water-only background at FIO2 in the range of 12 to 4%. The SO2 values calculated from the water-only background are higher than those of SaO2 at FIO2 in the range of 10 to 4%. However, SO2 values using the abs of the head with BET by FC are between those of SaO2 and SvO2 over the whole range of FIO2. Therefore, abs of the head with BET by FC is more useful for estimation of the absolute values of oxyHb and deoxyHb of the piglet head.

We have employed near-infrared optical methods to measure noninvasively the dynamics of muscle blood flow and oxygen saturation (StO2) during cuff occlusion and plantar flexion exercise. Relative muscle oxygen consumption (rVO2) was also computed from these data. Diffuse correlation spectroscopy provides information about blood flow, and diffuse reflectance spectroscopy provides information about blood oxygenation. Ten healthy subjects and one patient with peripheral arterial disease (PAD) were studied during 3-min arterial cuff occlusion of arm and leg, and during 1-min plantar flexion exercise. Signals from different layers (cutaneous tissues and muscles) during cuff occlusion were differentiated, revealing strong hemodynamic responses from muscle layers. During exercise in healthy legs, the observed ~4.7 fold increase in relative blood flow (rBF) was significantly lower than the corresponding increase in rVO2 (~7 fold). The magnitudes of rBF and rVO2 during exercise in the PAD patient were ~1/2 of the healthy controls, and the StO2 recovery time was twice that of the controls. The hybrid instrument improves upon current technologies for measuring muscle responses by simultaneously measuring rBF and StO2. The instrument thus provides a method for evaluation of microcirculation and muscle metabolism in patients with vascular diseases.

We investigate the optical properties of the brain in 23 neonates in vivo using a frequency domain near-infrared spectroscopy (NIRS). In this study, a calibration procedure is employed to determine the absorption and reduced scattering coefficients with single source-detector separation. The absorption coefficients of the infant foreheads are lower than the values reported in adults. A large intersubject variation in the reduced scattering coefficients is also demonstrated. Furthermore, physiological parameters are derived from the absorption coefficients at two wavelengths (788 and 832 nm). The mean total hemoglobin concentration (THC) is 39.7±9.8 µM and the mean cerebral blood oxygen saturation (StO2) is 58.7±11.2%. Our preliminary results show that this bedside frequent domain NIRS could provide quantitative optical measurement of the infant brain.

In a continuing effort to develop a noninvasive means of monitoring glucose levels using the aqueous humor of the eye, a dual-wavelength system is developed to show that varying birefringence, similar to what is seen with a moving cornea, can be compensated. In this work, a dual-wavelength, closed-loop system is designed and a model is developed to extract the glucose concentration information. The system and model are tested using various concentrations of glucose in a birefringent test cell subject to motion artifact. The results show that for a static, nonmoving sample, glucose can be predicted to within 10 mg/dl for the entire physiologic range (0 to 600 mg/dl) for either laser wavelength (523 or 635 nm). In the presence of moving birefringence, each individual wavelength produces standard errors on the order of a few thousand mg/dL. However, when the two wavelengths are combined into the developed model, this error is less than 20 mg/dL. The approach shows that multiple wavelengths can be used to drastically reduce the error in the presence of a moving birefringent sample and thus may have the potential to be used to noninvasively monitor glucose levels in vivo in the presence of moving corneal birefringence.

We present an initial study on applying genetic algorithms (GA) to retrieve human skin optical properties using visual reflectance spectroscopy (VRS). A three-layered skin model consisting of 13 parameters is first used to simulate skin and, through an analytical model based on optical diffusion theory, we study their independent effects on the reflectance spectra. Based on a preliminary analysis, nine skin parameters are chosen to be fitted by GA. The fitting procedure is applied first on simulated reflectance spectra with added white noise, and then on measured spectra from normal and port wine stain (PWS) human skin. A normalized residue of less than 0.005 is achieved for simulated spectra. In the case of measured spectra from human skin, the normalized residue is less than 0.01. Comparisons between applying GA and manual iteration (MI) fitting show that GA performed much better than the MI fitting method and can easily distinguish melanin concentrations for different skin types. Furthermore, the GA approach can lead to a reasonable understanding of the blood volume fraction and other skin properties, provided that the applicability of the diffusion approximation is satisfied.

Optical technologies, such as reflectance and fluorescence spectroscopy, have shown the potential to provide improved point-of-care detection methods for cervical neoplasia that are sensitive, specific, and cost-effective. Our specific goals are to analyze the diagnostic potential of reflectance and fluorescence spectra, alone and in combination, to discriminate normal and precancerous cervical tissue in vivo and to identify which classification features contain significant diagnostic information. Reflectance spectra are measured at four source-detector separations and fluorescence emission spectra are measured at 16 excitation wavelengths, from 324 sites in 161 patients. These 20 spectral features are permuted in all possible combinations of one, two, and three; and classification algorithms are developed to evaluate the diagnostic performance of each combination. Algorithms based on fluorescence spectra alone yield better diagnostic performance than those based on reflectance spectra alone. The combination of fluorescence and reflectance do not significantly improve diagnostic performance compared to fluorescence alone, except in the case of discriminating high-grade precancers from columnar normal tissue. In general, fluorescence emission spectra at 330- to 360-nm and 460- to 470-nm excitation provide the best diagnostic performance for separating all pairs of tissue categories.

We explore the effects of the illumination and collection geometry on optical spectroscopic diagnosis of breast cancer. Fluorescence and diffuse reflectance spectroscopy in the UV-visible spectral range are made with a multiseparation probe at three illumination-collection separations of 735, 980, and 1225 µm, respectively, from 13 malignant and 34 nonmalignant breast tissues. Statistical analysis is carried out on two types of data inputs: (1) the fluorescence and diffuse reflectance spectra recorded at each of the three illumination-collection separations and (2) the integrated fluorescence (at each excitation wavelength) or diffuse reflectance over the entire spectrum at all three illumination-collection separations. The results show that using the integrated fluorescence intensities recorded at a single excitation wavelength at all three illumination-collection separations can discriminate malignant from nonmalignant breast tissues with similar classification accuracy to that using spectral data measured at several excitation wavelengths with a single illumination-collection separation. These findings have significant implications with respect to the design of an optical system for breast cancer diagnosis. Examining the intensity attenuation at a single wavelength rather than spectral intensities at multiple wavelengths can significantly reduce the measurement and data processing time in a clinical setting as well as the cost and complexity of the optical system.

We describe what is, to the best of our knowledge, the first pilot study of coregistered tomographic x-ray and optical breast imaging. The purpose of this pilot study is to develop both hardware and data processing algorithms for a multimodality imaging method that provides information that neither x-ray nor diffuse optical tomography (DOT) can provide alone. We present in detail the instrumentation and algorithms developed for this multimodality imaging. We also present results from our initial pilot clinical tests. These results demonstrate that strictly coregistered x-ray and optical images enable a detailed comparison of the two images. This comparison will ultimately lead to a better understanding of the relationship between the functional contrast afforded by optical imaging and the structural contrast provided by x-ray imaging.

We report the application of a support vector machine (SVM) for the development of diagnostic algorithms for optical diagnosis of cancer. Both linear and nonlinear SVMs have been investigated for this purpose. We develop a methodology that makes use of SVM for both feature extraction and classification jointly by integrating the newly developed recursive feature elimination (RFE) in the framework of SVM. This leads to significantly improved classification results compared to those obtained when an independent feature extractor such as principal component analysis (PCA) is used. The integrated SVM-RFE approach is also found to outperform the classification results yielded by traditional Fisher's linear discriminant (FLD)-based algorithms. All the algorithms are developed using spectral data acquired in a clinical in vivo laser-induced fluorescence (LIF) spectroscopic study conducted on patients being screened for cancer of the oral cavity and normal volunteers. The best sensitivity and specificity values provided by the nonlinear SVM-RFE algorithm over the data sets investigated are 95 and 96% toward cancer for the training set data based on leave-one-out cross validation and 93 and 97% toward cancer for the independent validation set data. When tested on the spectral data of the uninvolved oral cavity sites from the patients it yielded a specificity of 85%.

An optoacoustic detection method suitable for depth profiling of optical absorption of layered or continuously varying tissue structures is presented. Detection of thermoelastically induced pressure transients allows reconstruction of optical properties of the sample to a depth of several millimeters with a spatial resolution of 24 µm. Acoustic detection is performed using a specially designed piezoelectric transducer, which is transparent for optical radiation. Thus, ultrasonic signals can be recorded at the same position the tissue is illuminated. Because the optoacoustical sound source is placed in the pulsed-acoustic near field of the pressure sensor, signal distortions commonly associated with acoustical diffraction are eliminated. Therefore, the acoustic signals mimic exactly the depth profile of the absorbed energy. This is illustrated by imaging the absorption profile of a two-layered sample with different absorption coefficients, and of a dye distribution while diffusing into a gelatin phantom.

Pterygia, caused by fibrovascular growth of conjunctiva, are a common ophthalmic disease. However, the molecular composition of pterygium tissue has not been completely understood, and therefore the aim of this study is to investigate the spectroscopic differences between normal human bulbar conjunctiva and human pterygium tissue using a confocal Raman system. The high signal-to-noise ratio spectra from pterygium and conjunctiva tissue were obtained by this technique without any sample preparation and the time of detection required less than 3 min. Comparing Raman spectra of two types of tissue, there are obvious changes, including intensity decrease at ~1585 cm–1 and intensity increase at ~1748, 1156, and 1521 cm–1 with the lesion of conjunctiva. Additionally, the amide I vibrational mode of proteins in conjunctiva is significantly different than that in pterygium. The use of pathology, immunology, and the peroxidation of the lipids in conjunction with the Raman results indicate that the presence of additional elastic fibers, mast cells, and lymphocytes in pterygium, as compared with normal bulbar conjunctiva, have fewer unsaturated fat acids. The present study demonstrates that Raman spectroscopy can be potentially applied to diagnose pterygia clinically.

We present functional imaging of the concentration of a photodynamic therapy (PDT)-related dye in scattering tissue phantoms based on spatially resolved measurements of optical properties through spectroscopic optical coherence tomography (OCT). Expressions for the OCT signal are developed, enabling estimation of depth-resolved sample optical properties. Based on these expressions, we discuss speckle statistics and speckle correlations of the OCT signal. Speckle noise reduction is performed by spatial filtering and is used to improve accuracy in the estimated optical properties at the expense of spatial resolution. An analytic expression for the precision in the estimated optical properties is derived. This expression shows that axial filtering, and thereby a reduction of axial resolution, gives a larger improvement in precision compared to the same filtering and reduction in the transversal resolution. It also shows that imaging with a shorter coherence length, or a larger numerical aperture, improves precision when the filter length determines the spatial resolution. Good agreement is obtained between experimentally determined and theoretically predicted variance in the estimated attenuation coefficients and dye concentration. Finally, we present guidelines for spectroscopic OCT systems for concentration imaging and discuss application of the method to more realistic phantoms and tissue.

An active, hardware-based retinal tracker is integrated with a clinical optical coherence tomography (OCT) system to investigate the effects of stabilization on acquisition of high-resolution retinal sections. The prototype retinal tracker locks onto common fundus features, detects transverse eye motion via changes in feature reflectance, and positions the OCT diagnostic beam to fixed coordinates on the retina with mirrors driven by a feedback control loop. The system is tested in a full clinical protocol on subjects with normal and glaucomatous eyes. Experimental analysis software is developed to coalign and coadd multiple fundus and OCT images and to extract quantitative information on the location of structures in the images. Tracking is highly accurate and reproducible on all but one subject, resulting in the ability to scan the same retinal location continually over long periods of time. The results show qualitative improvement in 97% of coadded OCT scans and a reduction in the variance of the position of the optic disc cup edge to less than 1 pixel (<60 µm). The tracking system can be easily configured for use in research on ultra-high-resolution OCT systems for advanced image modalities. For example, tracking will enable very high density 3-D scans of the retina, which are susceptible to eye motion artifacts even for new high-speed systems.