Raman spectroscopy is used to detect glutamate in the eye. Glutamate, a by-product of nerve cell death, is an indicator of glaucoma and diabetic retinopathy. The Raman spectra of ex vivo whole porcine eyes and individual components (lens, cornea, vitreous) are measured and characterized. Monosodium glutamate is injected into the eyes to simulate disease conditions, and the contribution to the Raman spectrum due to the presence of glutamate is identified. The Raman spectra from the native eye is dominated by vibrational modes from proteins in the lens. An optical system is designed to optimize collection of signal from the vitreous, where the glutamate is located, and reduce the Raman from the lens. Two vibrational fingerprints of monosodium glutamate are detected at 1369 and 1422 cm^–1, although the concentrations are much above physiological concentrations.

Hemoglobin (Hb) in single erythrocytes (red blood cells), adsorbed on polylysine-coated glass surfaces, was studied using resonance Raman spectroscopy and global Raman imaging. The erythrocytes were found to be sensitive to both surface adsorption and laser illumination. Substrate-dependent changes of the cell membrane shape were observed immediately after cell adsorption, while a photo-induced increase of fluorescence was observed for visible excitation (λ=514.5 nm). Concurrent changes in Raman spectra revealed a conversion of oxy-Hb (2+) to the inactive met-Hb state (3+). These effects severely complicate the interpretation of Raman images. However, at a low accumulated photon dose, the preparation method enabled the recording of Raman spectra during the oxygenation cycle of a single erythrocyte in buffer, which illustrates the feasibility of Raman investigations of functional cells in in-vivo environments.

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm^–1) and a decrease in reducible cross-links (1690 cm^–1). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.

Infrared (IR) microscopic imaging is used, in a series of proof-of-principle experiments to map the spatial distribution of two penetration enhancers, dimethylsulphoxide (DMSO) and propylene glycol, in skin. The current instrumentation utilizes a 64×64 array of IR detectors imaged at the focal plane of an IR microscope, each collecting a complete mid-infrared spectrum of the skin section on each pass of the interferometer. The spatial area sampled by each element in the array is ~6.3×6.3 μm. Any spectral parameter (e.g., arising from lipid or protein vibrational modes of the endogenous tissue or the exogenous component) may be quantitatively analyzed across the entire array of 4096 spectra, thereby generating an IR spectroscopic image of that particular parameter throughout the sample. The images directly reveal the spatial heterogeneity of the protein and lipid distributions. In transverse slices of skin, the depth dependencies of the spatial distribution of triglyceride and protein have been monitored, and compared to those of the exogenous penetration enhancers. Images of both DMSO and propylene glycol suggest that each penetrates the skin to a depth of at least 1 mm (under our experimental protocols), and reveals a spatial distribution that is essentially coincident with the protein constituents of the skin. These results demonstrate that IR microscopic imaging has great potential for mechanistic studies of topical, dermal, and transdermal delivery.

We determine temperature effect on the absorption and reduced scattering coefficients (μ_a and μ's) of human forearm skin. Optical and thermal simulation data suggest that μ_a and μ's are determined within a temperature-controlled depth of ≈ 2 mm. Cutaneous μ's change linearly with temperature. Change in μ_a was complex and irreversible above body normal temperatures. Light penetration depth δ in skin increased on cooling, with considerable person-to-person variations. We attribute the effect of temperature on μ's to change in refractive index mismatch, and its effect on μ_a to perfusion changes. The reversible temperature effect on μ's was maintained during more than 90 min. contact between skin and the measuring probe, where temperature was modulated between 38 and 22°C for multiple cycles While temperature modulated μ's instantaneously and reversibly, μ_a exhibited slower response time and consistent drift. There was a statistically significant upward drift in μ_a and a mostly downward drift in μ's over the contact period. The drift in temperature-induced fractional change in μ's was less statistically significant than the drift in μ's. Δμ's values determined under temperature modulation conditions may have less nonspecific drift than μ's which may have significance for noninvasive determination of analytes in human tissue.

Accurate data on in vivo tissue optical properties in the ultraviolet A (UVA) to visible (VIS) range are needed to elucidate light propagation effects and to aid in identifying safe exposure limits for biomedical optical spectroscopy. We have performed a preliminary study toward the development of a diffuse reflectance system with maximum fiber separation distance of less than 2.5 mm. The ultimate objective is to perform endoscopic measurement of optical properties in the UVA to VIS. Optical property sets with uniformly and randomly distributed values were developed within the range of interest: absorption coefficients from 1 to 25 cm^–1 and reduced scattering coefficients from 5 to 25 cm^–1. Reflectance datasets were generated by direct measurement of Intralipid-dye tissue phantoms at λ = 675 nm and Monte Carlo simulation of light propagation. Multivariate calibration models were generated using feed-forward artificial neural network or partial least squares algorithms. Models were calibrated and evaluated using simulated or measured reflectance datasets. The most accurate models developed—those based on a neural network and uniform optical property intervals—were able to determine absorption and reduced scattering coefficients with root mean square errors of ±2 and ±3 cm^–1, respectively. Measurements of ex vivo bovine liver at 543 and 633 nm were within 5 to 30% of values reported in the literature. While our technique for determination of optical properties appears feasible and moderately accurate, enhanced accuracy may be achieved through modification of the experimental system and processing algorithms.

Light scattering is used to monitor the dynamics and energy thresholds of laser-induced structural alterations in biopolymers due to irradiation by a free electron laser (FEL) in the infrared (IR) wavelength range 2.2 to 8.5 μm. Attenuated total reflectance (ATR) Fourier-transform IR (FTIR) spectroscopy is used to examine infrared tissue absorption spectra before and after irradiation. Light scattering by bovine and porcine cartilage and cornea samples is measured in real time during FEL irradiation using a 650-nm diode laser and a diode photoarray with time resolution of 10 ms. The data on the time dependence of light scattering in the tissue are modeled to estimate the approximate values of kinetic parameters for denaturation as functions of laser wavelength and radiant exposure. We found that the denaturation threshold is slightly lower for cornea than for cartilage, and both depend on laser wavelength. An inverse correlation between denaturation thresholds and the absorption spectrum of the tissue is observed for many wavelengths; however, for wavelengths near 3 and 6 μm, the denaturation threshold does not exhibit the inverse correlation, instead being governed by heating kinetics of tissue. It is shown that light scattering is useful for measuring the denaturation thresholds and dynamics for different biotissues, except where the initial absorptivity is very high.

The goal of the work is to experimentally verify Monte Carlo modeling of fluorescence and diffuse reflectance measurements in turbid, tissue phantom models. In particular, two series of simulations and experiments, in which one optical parameter (absorption or scattering coefficient) is varied while the other is fixed, are carried out to assess the effect of the absorption coefficient (μ_a) and scattering coefficient (μ_s) on the fluorescence and diffuse reflectance measured from a turbid medium. Moreover, simulations and experiments are carried out for several fiber optic probe geometries that are designed to sample small tissue volumes. Additionally, a group of conversion expressions are derived to convert the optical properties and fluorescence quantum yield measured from tissue phantoms for use in Monte Carlo simulations. The conversions account for the differences between the definitions of the absorption coefficient and fluorescence quantum yield of fluorophores in a tissue phantom model and those in a Monte Carlo simulation. The results indicate that there is good agreement between the simulated and experimentally measured results in most cases. This dataset can serve as a systematic validation of Monte Carlo modeling of fluorescent light propagation in tissues. The simulations are carried out for a wide range of absorption and scattering coefficients as well as ratios of scattering coefficient to absorption coefficient, and thus would be applicable to tissue optical properties over a wide wavelength range (UV-visible/near infrared). The fiber optic probe geometries that are modeled in this study include those commonly used for measuring fluorescence from tissues in practice.

Developing fiber optic probe geometries to selectively measure fluorescence spectra from different sublayers within human epithelial tissues will potentially improve the endogenous fluorescence contrast between neoplastic and nonneoplastic tissues. In this study, two basic fiber optic probe geometries, which are called the variable aperture (VA) and multidistance (MD) approaches, are compared for depth-resolved fluorescence measurements from human cervical epithelial tissues. The VA probe has completely overlapping illumination and collection areas with variable diameters, while the MD probe employs separate illumination and collection fibers with a fixed separation between them. Monte Carlo simulation results show that the total fluorescence detected is significantly higher for the VA probe geometry, while the probing depth is significantly greater for the MD probe geometry. An important observation is that the VA probe is more sensitive to the epithelial layer, while the MD probe is more sensitive to the stromal layer. The effect of other factors, including numerical aperture (NA) and tissue optical properties on the fluorescence measurements with VA and MD probe geometries, are also evaluated. The total fluorescence detected with both probe geometries significantly increases when the fiber NA is changed from 0.22 to 0.37. The sensitivity to different sublayers is found to be strongly dependent on the tissue optical properties. The simulation results are used to design a simple fiber optic probe that combines both the VA and MD geometries to enable fluorescence measurements from the different sublayers within human epithelial tissues.

We compared light-induced fluorescence (LIF) to nominal injected drug dose for predicting the depth of necrosis response to photodynamic therapy (PDT) in a murine tumor model. Mice were implanted with radiation-induced fibrosarcoma (RIF) and were injected with 0, 5, or 10 mg/kg Photofrin. 630-nm light (30 J/cm², 75 mW/cm²) was delivered to the tumor after 24 hours. Fluorescence emission (λ_excitation = 545 nm, λ_emission = 628 nm) from the tumor was measured. The LIF data had less scatter than injected drug dose, and was found to be at least as good as an injected drug dose for predicting the depth of necrosis after PDT. Our observations provide further evidence that fluorescence spectroscopy can be used to quantify tissue photosensitizer uptake and to predict PDT tissue damage.

Depth and radius of regions interrogated by cardiac optical mapping with a laser beam depend on photon travel inside the heart. It would be useful to limit the range of depth and radius interrogated. We modeled the effects of a condensing lens to concentrate laser light at a target depth inside the heart, and near infrared excitation to increase penetration and produce two-photon absorption. A Monte Carlo simulation that incorporated a 0.55-NA lens, and absorption and scattering of 1064- or 488-nm laser light in 3-D cardiac tissue indicated the distribution of excitation fluence inside the tissue. A subsequent simulation incorporating absorption and scattering of transmembrane voltage-sensitive fluorescence (wavelength 669 nm) indicated locations from which fluorescence photons exiting the tissue surface originated. The results indicate that mapping at depths up to 300 μm in hearts can provide significant improvement in localization over existing cardiac optical mapping. The estimated interrogation region is sufficiently small to examine cardiac events at a cellular or subcellular scale and may allow mapping at various depths in the heart.

Speckle, the dominant factor reducing image quality in optical coherence tomography (OCT), limits the ability to identify cellular structures that are essential for diagnosis of a variety of diseases. We describe a new high-speed method for implementing angular compounding by path length encoding (ACPE) for reducing speckle in OCT images. By averaging images obtained at different incident angles, with each image encoded by path length, ACPE maintains high-speed image acquisition and requires minimal modifications to OCT probe optics. ACPE images obtained from tissue phantoms and human skin in vivo demonstrate a qualitative improvement over traditional OCT and an increased SNR that correlates well with theory.

We use a modified Mach-Zehnder interferometer to measure surface displacement resulting from the thermoelastic response of a target to the absorption of a short laser pulse with axial and temporal resolutions of 0.1 nm and 3 ns, respectively. These measurements are used in conjunction with a solution to the thermoelastic wave equation and a nonlinear optimization algorithm to extract optical attenuation depth. We demonstrate the ability to determine the effective optical attenuation depth of homogeneous targets with either diffuse or specular reflecting surfaces with a precision of 4% for attenuation depths spanning 0.1 to 2 mm.

We introduce a minimally invasive technique for optoacoustic imaging of turbid media using optical interferometric detection of surface displacement produced by thermoelastic stress transients. The technique exploits endogenous or exogenous optical contrast of heterogeneous tissues and the low attenuation of stress wave propagation to localize and image subsurface absorbers in optically turbid media. We present a system that utilizes a time-resolved high-resolution interferometer capable of angstrom-level displacement resolution and nanosecond temporal resolution to detect subsurface blood vessels within model tissue phantoms and a human forearm in vivo.

Optoacoustic imaging was used for ophthalmic imaging, especially of the ciliary body region, which is of interest in the treatment of glaucoma. The different tissue structures below the sclera of porcine and rabbit eyes in vitro could be differentiated up to a depth of more than 1.5 mm. Based on the optoacoustic signals, two-dimensional tomographic images could be generated for visualization of this region in a B-scan mode. In addition, changes during the coagulation process could be measured in real time, allowing the development of online control mechanisms for cyclophotocoagulation, which is an important therapy of glaucoma.

We present a semianalytical technique to calculate the temperature in homogeneous nonperfused tissue during subablative laser heating. Analytical expressions of the temperature distribution in time and space are provided for collimated beams with Gaussian and top-hat intensity distributions perpendicularly incident on a finite tissue slab. The temperature distribution produced with a collimated Gaussian beam is the triple sum of the product of four functions of separate variables. The semianalytical technique can be used to rapidly calculate the temperature in laser-irradiated tissue at any point in time and space. The model was used to estimate the corneal temperature during pulsed holmium:YAG laser thermokeratoplasty with various boundary conditions at the anterior and posterior corneal surface. The model demonstrates that the corneal temperature during laser thermokeratoplasty (LTK) with a pulsed Ho:YAG laser may be sufficient to induce superficial vaporization of epithelial cells and local thermal damage to the endothelium. The calculations show that convection at the anterior corneal surface does not have a significant effect on the corneal temperature distribution, but that a better knowledge of the cooling effect of the aqueous is required to better estimate the corneal temperature distribution during LTK.

Laser technology has been studied as a potential replacement to the conventional dental drill. However, to prevent pulpal cell damage, information related to the safety parameters using high-power lasers in oral mineralized tissues is needed. In this study, the heat distribution profiles at the surface and subsurface regions of human dentine samples irradiated with a Nd:YAG laser were simulated using Crank-Nicolson's finite difference method for different laser energies and pulse durations. Heat distribution throughout the dentin layer, from the external dentin surface to the pulp chamber wall, were calculated in each case, to investigate the details of pulsed laser-hard dental tissue interactions. The results showed that the final temperature at the pulp chamber wall and at the dentin surface are strongly dependent on the pulse duration, exposure time, and the energy contained in each pulse.

There are unresolved clinical problems that require the provision of accurate 3-D images of tissue structures such as teeth. In particular, measurements of dental enamel thickness are necessary to quantify problems associated with enamel erosion, yet currently there is no nondestructive method to obtain this information. We present a method that relies on the use of pulsed terahertz radiation to gain 3-D information from dental tissues. We discuss results from 14 samples and demonstrate that we can reliably and accurately quantify enamel thickness. We show that in a series of 22 surfaces, we can image pertinent subsurface features 91% of the time. Example images are shown where structures in teeth at depth are rendered accurate to within 10 µm. We discuss issues that arise using this imaging method and propose ways in which it could be used in clinical practice.

A near-infrared (NIR) imaging system is evaluated as a diagnostic clinical tool to image total hemoglobin concentration and oxygen saturation within tissue. Calibration of this type of system requires measurement of the response at each detector and source location from a homogeneous tissue-simulating phantom. The effect of using calibration phantoms of varying composition, size, and optical properties is examined to determine how it affects the overall image accuracy. All of the calibration phantoms investigated result in accurate reconstruction of absorbing heterogeneities due to increased blood concentration with less than 4% standard deviation. Images from a patient with a biopsy-confirmed ductal carcinoma are also evaluated and found to be insensitive to the choice of calibration object, with only 1% variation between images generated with different calibration objects. The tumor total hemoglobin contrast is approximately 240% higher than the average total hemoglobin concentration in contralateral breast. Soft calibration phantoms, which mimic the elastic properties of human breast tissue, are also considered and found to diminish positioning errors in the fibers relative to the actual breast exam, thereby reducing the artifacts in the periphery of the reconstructed image.

This PDF file contains the errata for “JBO Vol. 8 Issue 02 Paper 1563261” for JBO Vol. 8 Issue 02