We present simultaneous measurement of enhancement kinetics of an optical and a magnetic resonance (MR) contrast agent in a small animal breast tumor model (R3230 ac) using a combined MR-diffuse optical tomographic (MR-DOT) imaging system. A mixture of a small molecular-weight MR contrast agent gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA) and a large molecular-weight optical contrast agent indocyanine green (ICG) was administered intravenously for multimodal dynamic imaging. Coregistration of optical and MR images was accomplished using agar-water–based markers. Using T₂ and dynamic T₁ weighted MR images, we divided the entire tumor into two regions of interest (ROI): a viable and a nonviable region. The absorption enhancements in the ROIs were calculated. An enhancement of the ICG was observed in the viable region. On the contrary, there was a lower enhancement in the nonviable region.

We report on a new optics design for an optical coherence tomography (OCT) balloon imaging catheter. The design involves a miniature compound gradient-index (GRIN) rod lens, which consists of a fiber optic mode-field reducer and relay rod lenses to achieve predictable high lateral resolution at a desired large working distance. The compound lens design significantly simplifies the engineering process for an OCT catheter and enables 3-D full circumferential cross sectional imaging of large luminal organs such as human esophagus. An as-designed OCT catheter is developed and demonstrated for real-time in vivo swine esophagus imaging in a 3-D spiral fashion.

We find for the first time that polarization mismatch of the sample and reference arms in optical-fiber-based optical coherence tomography (OCT) has critical effect on its depth resolution when the light source is partially polarized. When the polarization states of the two arms are matched, the measured point spread function (PSF) is almost identical to the theoretical prediction. When their polarization states are mismatched, the PSF can be so distorted that the depth resolution is degraded to several times the theoretical value. When we polarize the source light with a polarizer, then the degree of polarization (DOP) is unity, and the depth resolution becomes independent of the polarization mismatch. This discovery has fundamental importance for high-resolution OCT imaging of biological tissues. With DOP<1, the depth resolution can be quickly degraded by either birefringence or scattering in the sample. Adjusting polarization controllers can only improve the depth resolution at a certain depth in a sample if the polarization state of light changes along the depth. When DOP=1, uniform resolution along the depth of a sample can be achieved.

General-purpose computing on graphics processing units (GPGPU) is shown to dramatically increase the speed of Monte Carlo simulations of photon migration. In a standard simulation of time-resolved photon migration in a semi-infinite geometry, the proposed methodology executed on a low-cost graphics processing unit (GPU) is a factor 1000 faster than simulation performed on a single standard processor. In addition, we address important technical aspects of GPU-based simulations of photon migration. The technique is expected to become a standard method in Monte Carlo simulations of photon migration.

A hybrid optical device that uses a multimode fiber coupled to a tunable light source for illumination and a 2.4-mm photodiode for detection in contact with the tissue surface is developed as a first step toward our goal of developing a cost-effective, miniature spectral imaging device to map tissue optical properties in vivo. This device coupled with an inverse Monte Carlo model of reflectance is demonstrated to accurately quantify tissue absorption and scattering in tissue-like turbid synthetic phantoms with a wide range of optical properties. The overall errors for quantifying the absorption and scattering coefficients are 6.0±5.6 and 6.1±4.7%, respectively. Compared with fiber-based detection, having the detector right at the tissue surface can significantly improve light collection efficiency, thus reducing the requirement for sophisticated detectors with high sensitivity, and this design can be easily expanded into a quantitative spectral imaging system for mapping tissue optical properties in vivo.

Studying hemodynamic changes during early mammalian embryonic development is critical for further advances in prevention, diagnostics, and treatment of congenital cardiovascular (CV) birth defects and diseases. Doppler optical coherence tomography (OCT) has been shown to provide sensitive measurements of blood flow in avian and amphibian embryos. We combined Doppler swept-source optical coherence tomography (DSS-OCT) and live mouse embryo culture to analyze blood flow dynamics in early embryos. SS-OCT structural imaging was used for the reconstruction of embryo morphology and the orientation of blood vessels, which is required for calculating flow velocity from the Doppler measurements. Spatially and temporally resolved blood flow profiles are presented for the dorsal aorta and a yolk sac vessel in a 9.5-day embryo. We demonstrate that DSS-OCT can be successfully used for structural analysis and spatially and temporally resolved hemodynamic measurements in developing early mammalian embryos.

Lifetime imaging microscopy is a powerful tool to probe biological phenomena independent of luminescence intensity and fluorophore concentration. We describe time-resolved imaging of long-lifetime luminescence with an unmodified commercial laser scanning confocal/multiphoton microscope. The principle of the measurement is displacement of the detection pinhole to collect delayed luminescence from a position lagging the rasting laser beam. As proof of principle, luminescence from microspheres containing europium (Eu³⁺), a red emitting probe, was compared to that of short-lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of 1 Airy unit, the short-lifetime fluorescence of fluorescein, rhodamine and green microspheres disappeared much more rapidly than the long-lifetime phosphorescence of Eu³⁺ microspheres as the pinhole was repositioned in the lagging direction. In contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short- and long-lifetime luminescence. From measurements at different lag pinhole positions, a lifetime of 270 μs was estimated for the Eu³⁺ microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy.

We present a systematic experimental comparison of pulsed photothermal temperature profiling utilizing the customary spectral band of the InSb radiation detector (λ=3.0 to 5.6 μm) and a narrowed acquisition band (4.5 to 5.6 μm). We use custom tissue phantoms composed of agar gel layers separated by thin absorbing layers. The laser-induced temperature profiles are reconstructed within the customary monochromatic approximation, using a custom minimization algorithm. In a detailed numerical simulation of the experimental procedure, we consider several acquisition spectral bands with the lower wavelength limit varied between 3.0 and 5.0 μm (imitating application of different long-pass filters). The simulated PPTR signals contain noise with amplitude and spectral characteristics consistent with our experimental system. Both experimental and numerical results indicate that spectral filtering reduces reconstruction error and broadening of temperature peaks, especially for shallower and more complex absorbing structures. For the simulated PPTR system and watery tissues, numerical results indicate an optimal lower wavelength limit of 3.8 to 4.2 μm.

We present in vivo human total retinal blood flow measurements using Doppler Fourier domain optical coherence tomography (OCT). The scan pattern consisted of two concentric circles around the optic nerve head, transecting all retinal branch arteries and veins. The relative positions of each blood vessel in the two OCT conic cross sections were measured and used to determine the angle between the OCT beam and the vessel. The measured angle and the Doppler shift profile were used to compute blood flow in the blood vessel. The flows in the branch veins was summed to give the total retinal blood flow at one time point. Each measurement of total retinal blood flow was completed within 2 s and averaged. The total retinal venous flow was measured in one eye each of two volunteers. The results were 52.90±2.75 and 45.23±3.18 μl/min, respectively. Volumetric flow rate positively correlated with vessel diameter. This new technique may be useful in the diagnosis and treatment of optic nerve and retinal diseases that are associated with poor blood flow, such as glaucoma and diabetic retinopathy.

An investigation into the use of Raman optical tweezers to study urological cell lines is reported, with the ultimate aim of determining the presence of malignant CaP cells in urine and peripheral fluids. To this end, we trapped and analyzed live CaP cells (PC-3) and bladder cells (MGH-U1), because both prostate and bladder cells are likely to be present in urine. The laser excitation wavelength of 514.5 nm was used, with Raman light collected both in back- and forward-scattering geometric configurations. For the backscattering configuration the same laser was used for trapping and excitation, while for forward scattering a 1064 nm laser provided the trapping beam. Analysis of cell-diameter distributions for cells analyzed suggested normal distribution of cell sizes, indicating an unbiased cell-selection criterion. Principal components analysis afforded discrimination of MGH-U1 and PC-3 spectra collected in either configuration, demonstrating that it is possible to trap, analyze, and differentiate PC-3 from MGH-U1 cells using a 514.5 nm laser. By loading plot analysis, possible biomolecules responsible for discrimination in both configurations were determined. Finally, the effect of cell size on discrimination was investigated, with results indicating that separation is based predominantly on cell type rather than cell size.

The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging (DSI) at 9.4 T (b_max 7000 s/mm, 150-μm isotropic voxels), a method that derives the preferential diffusion of water/voxel, and high-throughput (10 fps) two-photon microscope (TPM). Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function (ODF) derived from the local diffusion (DSI) and 3-D structural autocorrelation (TPM), respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.

Radio-frequency (RF) tissue fusion is a novel method of tissue approximation that can seal tissue without the need for sutures or staples, based on the combined effects of heat and pressure on the apposed tissue surfaces. RF delivery must be controlled and optimized to obtain a reproducible, reliable seal. We use real-time optical measurements to improve understanding of the tissue modifications induced by RF fusion. The main macroscopic transformations are thermal denaturation and dehydration. Light propagation in tissue is a function of both and therefore should provide interesting insight into the dynamic of occurring phenomena. Quantification by continuous wave technique has proven challenging. We proposed an algorithm based on the measurement of the absolute transmittance of the tissue, making use of the modified Beer-Lambert law. The experimental method and the data algorithm are demonstrated by RF fusion of porcine small bowel. The proposed optical measurement modality is well adapted to modern surgical instrumentation used for minimally invasive procedures.

We introduce a multimodal facial color imaging modality that provides a conventional color image, parallel and cross-polarization color images, and a fluorescent color image. We characterize the imaging modality and describe the image analysis methods for objective evaluation of skin lesions. The parallel and cross-polarization color images are useful for the analysis of skin texture, pigmentation, and vascularity. The polarization image, which is derived from parallel and cross-polarization color images, provides morphological information of superficial skin lesions. The fluorescent color image is useful for the evaluation of skin chromophores excited by UV-A radiation. In order to demonstrate the validity of the new imaging modality in dermatology, sample images were obtained from subjects with various skin disorders and image analysis methods were applied for objective evaluation of those lesions. In conclusion, we are confident that the imaging modality and analysis methods should be useful tools to simultaneously evaluate various skin lesions in dermatology.

Age-related macular degeneration (AMD) is among the major concerns in ophthalmology, as it is the primary cause for irreversible blindness in developed countries. Nevertheless, there is poor understanding of the origins and mechanisms that trigger this important ocular disease. In common clinical pratice, AMD is monitored by autofluorescence imaging of the retinal pigment epithelial (RPE) cells through a confocal scanning laser ophthalmoscope. The RPE cells derive their dominant autofluorescence from the lipofuscin granules that accumulate in the cytoplasm with increasing age and disease. We explored a different approach to retinal RPE imaging using two-photon excited autofluorescence, offering intrinsic three-dimensional resolution, larger sensing depth and reduced photodamage compared to single-photon excited fluorescence ophthalmoscopy. A two-photon microscope, based on the architecture of a conventional scanning laser ophthalmoscope (HRT, Heidelberg Engineering, Germany), was designed for autofluorescence imaging on retina samples from postmortem human-donor eyes. We were able to visualize at video-rate speed single RPE lipofuscin granules, demonstrating the potential to develop this method toward clinical practice for patients with RPE-related retinal disease like AMD.

Attenuated total reflection Fourier transform infrared spectroscopic imaging combined with tape-stripping is an advantageous approach to map the depth penetration and lateral distribution of topically applied chemicals in Stratum corneum (SC) and the conformational order of SC lipids. Tape-stripping progressively removes layers of SC, and chemical imaging provides spatially resolved information on the chemical composition of both the newly exposed SC surface and of the tapes used for stripping. The procedure is rapid, minimally invasive, and does not necessitate cross-sectioning of the skin. This approach offers a simple and direct way to determine the distribution of exogenous volatile and non-volatile chemicals in SC as a function of the chemical composition of the formulation and time, and the conformational order of SC lipids in native and topically treated skin. The procedure described here is well suited to address questions of relevance for the areas of drug delivery, dermatology, and skin care.

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

An adaptive optics (AO) system was incorporated into a laser retinal exposure setup in order to correct for refractive error and higher-order aberrations of the nonhuman primate (NHP) eye during an in vivo retinal ED₅₀ measurement. Using this system, the ED₅₀ for a 100-ms, 532-nm small spot size exposure was measured to be 1.05 mJ total intraocular energy (TIE), a reduction of 22% from the value measured without aberration correction. The ED₅₀ for a 3.5-ns, 532-nm exposure was measured to be 0.51 μJ TIE, the lowest ED₅₀ reported for a ns-duration exposure. This is a reduction of 37% from the value measured without aberration correction and is a factor of only 2.6 higher than the maximum permissible exposure (MPE) for a 3.5-ns, visible wavelength small spot size exposure. The trend of in vitro measurements using retinal explants suggests that the in vivo ED₅₀ for small spot-size exposures could potentially be one order of magnitude smaller than the previously reported in vivo ED50. Distortion of the incident laser beam by ocular aberrations cannot fully explain the discrepancy between the in vivo measurements with no aberration correction and the in vitro results.

We present a Monte Carlo model to predict fluorescence spectra of the oral mucosa obtained with a depth-selective fiber optic probe as a function of tissue optical properties. A model sensitivity analysis determines how variations in optical parameters associated with neoplastic development influence the intensity and shape of spectra, and elucidates the biological basis for differences in spectra from normal and premalignant oral sites. Predictions indicate that spectra of oral mucosa collected with a depth-selective probe are affected by variations in epithelial optical properties, and to a lesser extent, by changes in superficial stromal parameters, but not by changes in the optical properties of deeper stroma. The depth selective probe offers enhanced detection of epithelial fluorescence, with 90% of the detected signal originating from the epithelium and superficial stroma. Predicted depth-selective spectra are in good agreement with measured average spectra from normal and dysplastic oral sites. Changes in parameters associated with dysplastic progression lead to a decreased fluorescence intensity and a shift of the spectra to longer emission wavelengths. Decreased fluorescence is due to a drop in detected stromal photons, whereas the shift of spectral shape is attributed to an increased fraction of detected photons arising in the epithelium.

Spatially resolved spectroscopy (SRS) allows the estimation of absolute tissue oxygen saturation, the ratio of oxygenated to total hemoglobin concentration, which may facilitate the comparison of results among patients. Eighty-two premature infants were included over two years. The cerebral tissue oxygenation index (c-TOI) was measured using NIRO 300 (Hamamatsu Photonics KK). c-TOI was measured at several positions in each infant. c-TOI varied over time, increasing in the first third and decreasing in the last third of the study period (p<10^-6). Two probes were used in the study, and a highly significant difference was found between these (p<10^-6). The mean difference was 8.5% (95%CI 5.4 to 11.6%). After correction for this difference, there was no variation over time. A conclusive explanation for the bias could not be identified. Since the study groups were well distributed, the bias had no influence on the results of our clinical study. We investigated an unexpected but highly significant probe-dependent bias in c-TOI with no conclusive explanation. Hence, comparisons of absolute TOI between groups of patients and among studies should be regarded with caution. A better strategy to detect potential instrumental problems will be useful in preventing biased c-TOI from occurring.

Voltage-sensitive dyes (VSDs) provide a spatially resolved optical read-out of electrical signals in excitable tissues. Several common fluorescent VSDs display electrochromic shifts of their emission spectra, making them suitable candidates for ratiometric measurements of transmembrane voltages. These advantages of VSDs are tempered by tissue-specific shifts to their fluorescence emission. In addition, the optimal electrochromic dye response occurs in wavelength bands distinct from the dye's maximal resting emission. This "action spectrum" can undergo tissue-specific shifts as well. We have developed a technique for in situ measurements of the action spectra of VSDs in intact excitable tissues. Fluorescence emission spectra of VSDs during action-potential depolarization were obtained within a single sweep of a spectrophotometer equipped with a change-coupled device (CCD) array detector. To resolve the subtle electrochromic shifts in voltage-induced dye emission, fluorescence emission spectra measured right before and during field-induced action-potential depolarization were averaged over about 100 trials. Removing white-noise contributions from the spectrometer's CCD detector/amplifier via low-pass filtering in Fourier space, the action spectra of all dyes could be readily determined.

Development, validation, and implementation of an analytical model to extract biologically and diagnostically relevant parameters from measured cervical tissue reflectance and fluorescence spectra are presented. Monte Carlo simulations of tissue reflectance are used to determine the relative contribution of the signal from the epithelium and stroma. The results indicate that the clinical probe used collects a majority of its reflectance signal from the stroma; therefore, a one-layer analytical model of reflectance is used. Two analytical approaches to calculate reflectance spectra are compared to Monte Carlo simulations, and a diffusion theory-based model is implemented. The model is validated by fitting spectra generated from Monte Carlo simulations and comparing the input and output parameters. Median agreement between extracted optical properties and input parameters is 10.6%. The reflectance model is used together with an analytical model of tissue fluorescence to extract optical properties and fluorophore concentrations from 748 clinical measurements of cervical tissue. A diagnostic algorithm based on these extracted parameters is developed and evaluated using cross-validation. The sensitivity/specificity of this algorithm relative to the gold standard of histopathology per measurement are 85/51%; this is comparable to accuracy reported in other studies of optical technologies for detection of cervical cancer and its precursors.

Angiogenesis is a dynamic process that requires an interaction of pro-and antiangiogenic factors. It is known that the cytokine leptin stimulates endothelial cell growth and angiogenesis, but further quantitative analysis is necessary to understand leptin angiogenic effects. The quail chorioallantoic membrane (CAM) assay has been used to study angiogenesis in vivo by focusing on morphometric parameters that quantify vascular complexity and density. We quantify the angiogenic activity of leptin using the CAM assay by digital morphometry and a computer-assisted image analysis to evaluate more precisely vessel length, diameter, branching, and tortuousity. CAM images are obtained from ex ovo cultures of E8-E9 quail embryos. MATLAB® and custom software are used for our analysis. The effects of leptin, vascular endothelial growth factor-165 (VEGF₁₆₅), and their corresponding neutralizing antibodies are compared. Our results show that CAM treated with leptin and VEGF₁₆₅ has a significant increase in vascular complexity and density. A corresponding decrease is observed using neutralizing antibodies. Notably, leptin induced more significant changes than VEGF in vessel length and tortuousity. Conversely, VEGF induced a greater increase in vessel branching than leptin. These results underscore the importance of using multiparametric quantitative methods to assess several aspects of angiogenesis and enable us to understand the proangiogenic effects of leptin.

The ability to quantify changes in cardiomyocyte and myosin volume across gestation and in response to intrauterine insults will lead to a better understanding of the link between low birth weight and an increased risk of heart disease in adult life. We present the use of second-harmonic generation (SHG) and two-photon excitation autofluorescence (TPEF) microscopy to image unstained isolated fetal cardiomyocytes. The simultaneous collection of these two images provides a wealth of information on the morphology of cardiomyocytes. The SHG signal provides high-contrast images of myosin filaments and the TPEF signal can be used to clearly visualize cell morphology. A potential issue may arise if SHG microscopy is performed exclusively due to the lack of sensitivity to distinguish between mononucleated and binucleated cardiomyocytes. However, TPEF microscopy has the ability to efficiently separate the two types of cardiomyocytes. In addition, quantitative analysis of the SHG and TPEF images enables quantification of myosin filament level and accurate determination of cell volume. In short, we demonstrate that advanced nonlinear optical microscopy can be used to answer key physiological questions in the early origins of adult health with increased accuracy and speed compared to previously used methods.

Accurate estimation of concentration changes in muscles by continuous wave near-IR spectroscopy for muscle measurements suffers from underestimation and crosstalk problems due to the presence of superficial skin and fat layers. Underestimation error is basically caused by a homogeneous medium assumption in the calculations leading to the partial volume effect. The homogeneous medium assumption and wavelength dependence of mean partial path length in the muscle layer cause the crosstalk. We investigate underestimation errors and crosstalk by Monte Carlo simulations with a three layered (skin-fat-muscle) tissue model for a two-wavelength system where the choice of first wavelength is in the 675- to 775-nm range and the second wavelength is in the 825- to 900-nm range. Means of absolute underestimation errors and crosstalk over the considered wavelength pairs are found to be higher for greater fat thicknesses. Estimation errors of concentration changes for Hb and HbO₂ are calculated to be close for an ischemia type protocol where both Hb and HbO₂ are assumed to have equal magnitude but opposite concentration changes. The minimum estimation errors are found for the 700/825- and 725/825-nm pairs for this protocol.

We performed multiwavelength photoacoustic (PA) measurement for extensive deep dermal burns in rats to monitor the healing process of the wounds. The PA signal peak at 532 nm, an isosbestic point for oxyhemoglobin (HbO₂) and deoxyhemoglobin (HHb), was found to shift to a shallower region of the injured skin tissue with the elapse of time. The results of histological analysis showed that the shift of the PA signal reflected angiogenesis in the wounds. Until 24 h postburn, PA signal amplitude generally increased at all wavelengths. We speculate that this increase in amplitude is associated with dilation of blood vessels within healthy tissue under the injured tissue layer and increased hematocrit value due to development of edema. From 24 to 48 h postburn, the PA signal showed wavelength-dependent behaviors; signal amplitudes at 532, 556, and 576 nm continued to increase, while amplitude at 600 nm, an HHb absorption-dominant wavelength, decreased. This seems to reflect change from shock phase to hyperdynamic state in the rat; in the hyperdynamic state, cardiac output and oxygen consumption increased considerably. These findings show that multiwavelength PA measurement would be useful for monitoring recovery of perfusion and change in local hemodynamics in the healing process of burns.

A laser-based method has been developed for experimentally probing single red blood cell (RBC) buckling and determining RBC membrane rigidity. Our method combines a liquid flow cell, fluorescence microscopy, and an optical-trap to facilitate simple measurements of the shear modulus and buckling properties of single RBCs, under physiological conditions. The efficacy of the method is illustrated by studying buckling behavior of normal and Plasmodium-infected RBCs, and the effect of Plasmodium falciparum–conditioned medium on normal, uninfected cells. Our simple method, which quantifies single-RBC deformability, may ease detection of RBC hematological disorders.

Fluorescence spectroscopy contains diagnostic information about the lung biochemistry and morphology, including tissue optical properties and fluorophores. However, the fluorophore information is generally masked by the optical properties of the tissue, which complicates the evaluation of their role in lung-cancer detection. In this work, we have developed a method for extracting the intrinsic fluorescence spectra from the endoscopic measurements of the combined fluorescence and reflectance spectra. Principle components and classification analysis was performed to evaluate the diagnostic potential of the extracted intrinsic fluorescence spectra from in vivo combined fluorescence and reflectance spectral measurements. We evaluated the diagnostic sensitivity and specificity of both the intrinsic fluorescence and the fluorescence spectra. The results showed that the intrinsic fluorescence spectra contain significant diagnostic information that had been masked by the lung optical properties. We have also found that the intrinsic fluorescence has improved the specificity for endobronchial-cancer detection, although with a slight decrease in the detection sensitivity, when compared to the fluorescence spectra. This may indicate that intrinsic fluorescence analysis could be used to improve the diagnostic specificity of fluorescence spectroscopy and imaging.

Hyperglycemia and cortical spreading depression (CSD) are possible factors that worsen the outcome of ischemic stroke, and it is probable that there is a longterm cooperative effect of hyperglycemia and CSD on cerebral blood flow (CBF). Long-lasting and full-field observation of changes in CBF following CSD in vivo during acute hyperglycemia in rats might show whether this is the case. Here, we utilized laser speckle imaging to study influences of acute hyperglycemia on CBF at the level of individual vascular compartments for 3 h in normal rats and those with CSD. It is shown that there are extensive increases of CBF at the arteriole and parenchyma over the normal rat cortex during acute hyperglycemia, whereas there is no significant change in CBF at the venule. We also find that, at all vascular compartments, after the glucose administration there is a stepwise reduction of CBF following CSD, but after saline injection CBF following CSD is close to the baseline. Our results indicate that acute hyperglycemia could aggravate the severity of decrease in CBF following CSD, suggesting possible mechanisms by which hyperglycemia exacerbates cerebral damage after ischemic stroke.

We report the design of a novel laser line-triangulation laryngoscope for the quantitative visualization of the three-dimensional movements of human vocal folds during phonation. This is the first successful in vivo recording of the three-dimensional movements of human vocal folds in absolute values. Triangulation images of the vocal folds are recorded at the rate of 4000 fps with a resolution of 256×256 pixels. A special image-processing algorithm is developed to precisely follow the subpixel movements of the laser line image. Vibration profiles in both horizontal and vertical directions are calibrated and measured in absolute SI units with a resolution of ±50 µm. We also present a movie showing the vocal folds dynamics in vertical cross section.

We discuss the issue of separating contributions from mechanical and optical properties of a moderately scattering tissue phantom to the modulation depth (M) of intensity autocorrelation measured in an ultrasound-assisted optical tomography system using axial and transverse illuminations. For axial illumination, M is affected by both the displacement and absorption coefficient, more prominently by displacement. But transverse illumination has very little contribution from displacement of scattering centers. Since displacement is related to the elastic property of the insonified region, we show that there is a possibility of separating the contributions from elastic and optical properties of the insonified region using axial and transverse illuminations. The main conclusions of our study using moderately scattering phantoms are: 1. axial illumination is the best for mapping storage modulus inhomogeneities, but M is also affected by optical absorption; 2. transverse illumination is the best for mapping absorption inhomogeneities; and 3. for the practically relevant case of an inclusion with larger storage modulus and absorption, both illuminations produced large contrast in M. When the scattering coefficient is high, the angle dependence of illumination is lost and the present method is shown to fail to separate these contributions based on direction of illumination.

Depth analysis of the optic nerve head (ONH) in the retinal fundus is important for the early detection of glaucoma. In this study, we investigate an automatic reconstruction method for the quantitative depth measurement of the ONH from a stereo retinal fundus image pair. We propose a technique to obtain the depth value from the stereo retinal fundus image pair, which mainly consists of five steps: 1. cutout of the ONH region from the stereo retinal fundus image pair, 2. registration of the stereo image pair, 3. disparity measurement, 4. noise reduction, and 5. quantitative depth calculation. Depth measurements of 12 normal eyes are performed using the stereo fundus camera and the Heidelberg Retina Tomograph (HRT), which is a confocal laser-scanning microscope. The depth values of the ONH obtained from the stereo retinal fundus image pair were in good accordance with the value obtained using HRT (r=0.80±0.15). These results indicate that our proposed method could be a useful and easy-to-handle tool for assessing the cup depth of the ONH in routine diagnosis as well as in glaucoma screening.

We demonstrate noninvasive near-infrared diffuse optical spectroscopy (DOS) measurements of tissue hemoglobin contents that can track progressive reductions in central blood volume in human volunteers. Measurements of mean arterial blood pressure (MAP), heart rate (HR), stroke volume (SV), and cardiac output (Q) are obtained in ten healthy human subjects during baseline supine rest and exposure to progressive reductions of central blood volume produced by application of lower body negative pressure (LBNP). Simultaneous quantitative noninvasive measurements of tissue oxyhemoglobin (OHb), deoxyhemoglobin (RHb), total hemoglobin concentration (THb), and tissue hemoglobin oxygen saturation (S_tO₂) are performed throughout LBNP application using broadband DOS. As progressively increasing amounts of LBNP are applied, HR increases, and MAP, SV, and Q decrease (p<0.001). OHb, S_tO₂, and THb decrease (p<0.001) in correlation with progressive increases in LBNP, while tissue RHb remained relatively constant (p=0.378). The average fractional changes from baseline values in DOS OHb (fOHb) correlate closely with independently measured changes in SV (r²=0.95) and Q (r²=0.98) during LBNP. Quantitative noninvasive broadband DOS measurements of tissue hemoglobin parameters of peripheral perfusion are capable of detecting progressive reductions in central blood volume, and appear to be sensitive markers of early hypoperfusion associated with hemorrhage as simulated by LBNP.

Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.

We develop a new tomographic imaging reconstruction algorithm for a two-layer tissue structure. Simulations and phantom experiments show more accurate reconstruction of target optical properties compared with those results obtained from a semi-infinite tissue model for layered structures. This improvement is mainly attributed to the more accurate estimation of background optical properties and more accurate estimation of weight matrix for imaging reconstruction by considering the light propagation effect in the second layer. Clinical results of breast lesions are also presented to demonstrate the utility of this new imaging algorithm.

Transillumination breast spectroscopy (TiBS) uses nonionizing optical radiation to gain information about breast tissue morphological and structural properties. TiBS spectra are obtained from 232 women and compared to mammographic density (MD) quantified using Cumulus. The ability of TiBS to estimate MD is assessed using partial least-squares (PLS) regression methods, which requires TiBS spectra as input (X) and Cumulus MD as target (Y) data. Multiple PLS models are considered to determine the optimal processing technique(s) for the input (X) and target (Y) data. For each model, the association between TiBS estimated MD (Yˆ) and Cumulus MD (Y) is established using Spearman's rank correlation and linear regression analysis. The model that best estimates MD has the fewest assumptions regarding target (Y) and spectral (X) processing. The Spearman's correlation coefficient between predicted MD and Cumulus MD for this model is 0.88, with a regression slope (β) of 0.93 (95% CI 0.83–1.02) and an R² of 0.78. The approximation of individual MD was within 10% of Cumulus MD for the majority of women (80%), without stratification on age, body mass index (BMI), and menopausal status. TiBS provides an alternative to mammography assessed MD enabling frequent and earlier use of MD as a risk marker in preventive oncology.

Zinc oxide (ZnO-nano) and titanium dioxide nanoparticles (20 to 30 nm) are widely used in several topical skin care products, such as sunscreens. However, relatively few studies have addressed the subdermal absorption of these nanoparticles in vivo. We report on investigation of the distribution of topically applied ZnO in excised and in vivo human skin, using multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) and an energy-dispersive x-ray (EDX) technique to determine the level of penetration of nanoparticles into the sub-dermal layers of the skin. The good visualization of ZnO in skin achieved appeared to result from two factors. First, the ZnO principal photoluminescence at 385 nm is in the "quiet" spectral band of skin autofluorescence dominated by the endogenous skin fluorophores, i.e., NAD[P]H and FAD. Second, the two-photon action cross section of ZnO-nano [σ_ZnO^(TPEF)< ~0.26 GM; diameter, 18 nm] is high: ~500-fold of that inferred from its bulk third-order nonlinear susceptibility [Im χ_ZnO⁽³⁾], and is favorably compared to that of NAD[P]H and FAD. The overall outcome from MPM, SEM, and EDX studies was that, in humans in vivo, ZnO nanoparticles stayed in the stratum corneum (SC) and accumulated into skin folds and/or hair follicle roots of human skin. Given the lack of penetration of these nanoparticles past the SC and that the outermost layers of SC have a good turnover rate, these data suggest that the form of ZnO-nano studied here is unlikely to result in safety concerns.

We develop a method of coherent phase microscopy (CPM) for direct visualization of nonfixed, nonstained mammalian cells (both cultured cells and freshly isolated tumor biopsies) followed by computer-assisted data analysis. The major purpose of CPM is to evaluate the refractive properties of optically dense intracellular structures such as the nucleus and the nucleoli. In particular, we focus on quantitative real-time analysis of the nucleolar dynamics using phase thickness as an equivalent of optical path difference for optically nonhomogenous biological objects. Pharmacological inhibition of gene transcription leads to a dramatic decrease of the phase thickness of the nucleoli within the initial minutes of cell exposure. Furthermore, the acute depletion of intracellular ATP pool, depolymerization of microtubules and inhibition of DNA replication resulted in a rapid decrease of the nucleolar phase thickness. These optical effects were paralleled by segregation of nucleolar components as documented by electron microscopy. Thus, CPM detects early changes of nucleolar dynamics, in particular, the nucleolar segregation as part of general cellular response to cytotoxic stress, regardless of whether the nucleolus is or is not the primary target of the toxin. CPM is applicable for monitoring and quantitative analysis of the “nucleolar stress” in living mammalian cells.

Thin polymer etalons are demonstrated as high-frequency ultrasound sensors for three-dimensional (3-D) high-resolution photoacoustic imaging. The etalon, a Fabry-Perot optical resonator, consists of a thin polymer slab sandwiched between two gold layers. It is probed with a scanning continuous-wave (CW) laser for ultrasound array detection. Detection bandwidth of a 20-μm-diam array element exceeds 50 MHz, and the ultrasound sensitivity is comparable to polyvinylidene fluoride (PVDF) equivalents of similar size. In a typical photoacoustic imaging setup, a pulsed laser beam illuminates the imaging target, where optical energy is absorbed and acoustic waves are generated through the thermoelastic effect. An ultrasound detection array is formed by scanning the probing laser beam on the etalon surface in either a 1-D or a 2-D configuration, which produces 2-D or 3-D images, respectively. Axial and lateral resolutions have been demonstrated to be better than 20 μm. Detailed characterizations of the optical and acoustical properties of the etalon, as well as photoacoustic imaging results, suggest that thin polymer etalon arrays can be used as ultrasound detectors for 3-D high-resolution photoacoustic imaging applications.

In order to evaluate the impact of anatomy on the spectral properties of oral tissue, we used reflectance and fluorescence spectroscopy to characterize nine different anatomic sites. All spectra were collected in vivo from healthy oral mucosa. We analyzed 710 spectra collected from the oral cavity of 79 healthy volunteers. From the spectra, we extracted spectral parameters related to the morphological and biochemical properties of the tissue. The parameter distributions for the nine sites were compared, and we also related the parameters to the physical properties of the tissue site. k-Means cluster analysis was performed to identify sites or groups of sites that showed similar or distinct spectral properties. For the majority of the spectral parameters, certain sites or groups of sites exhibited distinct parameter distributions. Sites that are normally keratinized, most notably the hard palate and gingiva, were distinct from nonkeratinized sites for a number of parameters and frequently clustered together. The considerable degree of spectral contrast (differences in the spectral properties) between anatomic sites was also demonstrated by successfully discriminating between several pairs of sites using only two spectral parameters. We tested whether the 95% confidence interval for the distribution for each parameter, extracted from a subset of the tissue data could correctly characterize a second set of validation data. Excellent classification accuracy was demonstrated. Our results reveal that intrinsic differences in the anatomy of the oral cavity produce significant spectral contrasts between various sites, as reflected in the extracted spectral parameters. This work provides an important foundation for guiding the development of spectroscopic-based diagnostic algorithms for oral cancer.

Photodynamic therapy (PDT) is a promising cancer treatment that involves optical excitation of photosensitizers that promote oxygen molecules to the metastable O₂(a¹Δ) state (singlet oxygen). This species is believed to be responsible for the destruction of cancerous cells during PDT. We describe a fiber optic-coupled, pulsed diode laser-based diagnostic for singlet oxygen. We use both temporal and spectral filtering to enhance the detection of the weak O₂(a→X) emission near 1.27 µm. We present data that demonstrate real-time singlet oxygen production in tumor-laden rats with chlorin e6 and 5-aminolevulinic acid-induced protoporphyrin photosensitizers. We also observe a positive correlation between post-PDT treatment regression of the tumors and the relative amount of singlet oxygen measured. These results are promising for the development of the sensor as a real-time dosimeter for PDT.

We investigate the relationship between the fate and healing effect of transplanted mesenchymal stromal cells (MSCs) in a rat diabetic skin wound model. Rats are treated with streptozotocin to induce diabetic conditions. A full-thickness skin defect is surgically made on the head of diabetic rats, and covered with an artificial dermis impregnated with either bone marrow cells (BMCs) or bone-marrow-derived MSCs from firefly luciferase (luc) transgenic (Tg) rats. Wound healing is evaluated using planimetry and immunohistochemistry, and the fate of transplanted MSCs is determined using in-vivo luminescent imaging. The diabetic wound treated with MSCs-impregnated artificial dermis is significantly smaller than that treated with artificial dermis alone at 1 week postoperation. Photons of luc+ MSCs are detected at the transplanted site during healing (3 weeks), whereas those of luc+ MSCs are depleted only after 1 week postimplantation. Immunohistochemistry at the healing site treated with MSCs demonstrates that CD31+ vessels increase with expression of vascular endothelial growth factor, suggesting that MSCs accelerate angiogenesis. These findings suggest that transplanted MSCs could be retained at wound sites during the healing process in a diabetic rat model, and subsequently promote wound healing through angiogenesis.

Fluorescence recovery after photobleaching (FRAP) is a widely used method to measure diffusion. The technique is normally based on one-photon excitation, which limits diffusion to two dimensions due to extended photobleaching in the axial direction. Multiphoton excitation, on the other hand, creates a well-defined focal volume. In the present work, FRAP based on a scanning laser beam and two-photon excitation is used to measure diffusion of macromolecules in solution and gels, as well as in the extracellular matrix in multicellular spheroids and tumor tissue in dorsal chambers. The bleaching profile is determined experimentally in immobilized gels, and for small scanning areas (approximately twice the lateral radius of the laser beam) a Gaussian bleaching distribution is found. In addition, the bleaching profile is determined theoretically based on the convolution of the Gaussian point spread function and a circular scanning area. The diffusion coefficient is determined by fitting a mathematical model based on a Gaussian laser beam profile to the experimental recovery curve. The diffusion coefficient decreases with increasing complexity of the sample matrix and increasing the amount of collagen in the gels. The potential of using two-photon laser scanning microscopes for noninvasive diffusion measurements in tissue is demonstrated.

A time-domain optical method to evaluate the concentration (n), lifetime (τ), and depth (d) of a fluorescent inclusion is described by the complete analysis of the fluorescence temporal point-spread function (TPSF). The behavior of parameters in the fluorescence TPSF is explored, and we demonstrate the method with experimental data from a localized fluorescent inclusion in scattering media to recover images of n, τ, and d. The method has potential application for in vivo fluorescence imaging.

Tau is a microtubule associated protein that is localized to the axon in neurons. During pathological conditions, including frontotemporal dementia (FTD), a shift in tau isoforms occurs that leads to enhanced expression of a form of tau with four (rather than three) microtubule binding repeats; this has been postulated to alter microtubule structure. Second harmonic generation (SHG) is a technique that allows the visualization of intact microtubules in axons of living neurons without the need for labeling or fixing. We examined how the presence of exogenous tau influences SHG in living neurons. Our results show that the presence of tau significantly enhances SHG, specifically in neuronal axons, despite the presence of tau throughout the entire cell. Our data also suggest that the presence or absence of the fourth microtubule binding repeat does not significantly alter tau's ability to enhance SHG. These results provide evidence that SHG is a useful, noninvasive tool to study tau-microtubule interactions in axons; further, it appears that tau overexpression, rather than specific isoforms, is the major contributor to tau-induced changes in axonal microtubule SHG signal.

We report a novel postgrowth microwave heating implementation by selectively modifying hierarchical polystyrene (PS) bead substrates coated with gold (Au) films to effectively improve the surface-enhanced Raman scattering (SERS) effect on the analytes. The SERS signal of probe molecule rhodamine 6G (Rh 6G) on the microwave-treated Au–PS substrates can be improved by 10-fold, while the detection limit of Rh 6G in concentration can be enhanced by two orders of magnitude compared to the as-growth substrates. The high-quality SERS spectrum of saliva can also be acquired using the modified substrates, demonstrating the potential for the realization of the high-performance SERS substrates for biomedical applications.

Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy (HGM) can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations (SHG, THG) are virtual-state-transition-based systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1α, 2α, and 3α genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.

Differential path length spectroscopy (DPS) is a method of reflectance spectroscopy that utilizes a specialized fiber geometry to make the photon path length (τ) insensitive to variations in tissue optical properties over a wide range of absorption (μ_a) and total scattering (μ_s) coefficients, which are common within the ultraviolet/visible(UV/VIS) wavelength region. This study extends the description of τ to larger μ_a and smaller μ_s values, optical properties that are representative of the near-infrared region (NIR), a region where the DPS path length may be dependent on both coefficients. This study presents a novel empirical relationship between τ and the combined effect of both μ_a (range: 0.1–12 mm^-1) and μ_s (range: 1.5–42 mm^-1), anisotropy of 0.8, and is applicable to DPS probes containing a wide range of fiber diameters (range: 100–1000 μm). The results indicate that the simple empirical formula, including only one fitted parameter, is capable of accurately predicting over a wide range (r=0.985; range: 80–940 μm) and predictions are not biased versus μ_a or μ_s. This novel relationship is applicable to analysis of DPS measurements of tissue in both the UV/VIS and NIR wavelength regions and may provide information about the wavelength-specific tissue volume optically sampled during measurement.

Near-infrared (NIR) spectroscopy is used to quantify cerebral blood volume (CBV) as a marker of angiogenesis (formation of new blood vessels). Rats are exposed to chronic hypoxia for 3 weeks at half atmospheric pressure to stimulate angiogenesis, and second-differential NIR spectroscopy is used to quantify total cerebral hemoglobin before and after angiogenesis. The cerebral hemoglobin (from broadband NIR spectroscopy), and the large vessel hemoglobin and hematocrit (from blood samples), are used to derive values for the calculation of CBV. The total hemoglobin in brain is 46.6±1.9 μmol/l (mean±SD, n=5) preacclimation and increases by 72% postacclimation. CBV is initially 3.26±0.41% v/v and increases by 31% with acclimation. Each individual animal shows a measureable increase in CBV. This study indicates that NIR broadband spectroscopy can be used for repeated measurements of CBV and can be applied as a noninvasive method to study angiogenesis.

This PDF file contains the errata for “JBO Vol. 13 Issue 06 Paper 3054187” for JBO Vol. 13 Issue 06