Objective: At 380 nm excitation, cervical tissue fluorescence spectra demonstrate characteristic changes with both patient age and the presence of dysplasia. A Monte Carlo model was developed in order to quantitatively examine how intrinsic NADH and collagen fluorescence, in combination with tissue scattering and absorption properties, yield measured tissue spectra. Methods: Excitation-emission matrices were measured for live cervical cells and collagen gel phantoms. Fluorescence microscopy of fresh tissue sections was performed to obtain the location and density of fluorophores as a function of patient age and the presence of dysplasia. A Monte Carlo model was developed which incorporated measurements of fluorophore line shapes and spatial distributions. Results: Modeled spectra were consistent with clinical measurements and indicate that an increase in NADH fluorescence and decrease in collagen fluorescence create clinically observed differences between normal and dysplastic tissue spectra. Model predictions were most sensitive to patient age and epithelial thickness. Conclusions: Monte Carlo techniques provide an important means to investigate the combined contributions of multiple fluorophores to measured emission spectra. The approach will prove increasingly valuable as a more sophisticated understanding of in vivo optical properties is developed.

Immature and dysplastic cervical squamous epithelium whitens after the application of acetic acid during a colposcopic examination. The whitening process occurs visually over several minutes and subjectively discriminates between dysplastic and normal tissue. In this work, examples of the acetowhitening process are detailed in three ways: the color-imaged colposcopic appearance of the acetowhitening of high-grade cervical intraepithelial neoplasia (CIN 2/3), the kinetics of these reflectance patterns transformed to reduce noise in the signal, and a self-normalized green to red ratio measurement of the kinetics of these reflectance patterns. A total of six patients with biopsy confirmed CIN 2/3 were examined to obtain a set of timed images tracking the acetowhitening and the whitening-decay process over the course of 5–10 min. Regions of normal mature squamous epithelium within the same patients were also followed as an internal control. We determined that the temporal change over a 10 min time period in the ratio of green to red light intensities, taken from the respective color channels of the CCD, provides a reliable measure to clearly distinguish CIN 2/3 from normal cervical epithelium. This imaging and data normalization procedure may be applied to cervical lesions of different grades, to determine if a quantitative estimate provides predictive value during the colposcopic diagnosis.

The optical properties of melanin have been characterized for a number of laser wavelengths in the visible region. The index of refraction of melanin is measured by the conventional method of minimum deviation using a hollow quartz prism at these wavelengths. The inverse adding doubling method based on the diffusion approximation and radiative transport theory have been employed to determine the absorption, scattering, and scattering anisotropy coefficients of melanin from the measurements of diffuse transmission, diffuse reflection and collimated transmission using double integrating spheres. The results obtained by the use of inverse adding doubling method have been compared to the Monte Carlo simulation technique.

Optical low-coherence reflectometry (OLCR) was used as a noncontact method to measure the central corneal thickness of three patients intraoperatively during photorefractive keratectomy. Continuous on-line measurements were performed on the intact cornea immediately before the beginning of surgery, after the removal of the corneal epithelium, during laser tissue photoablation, and for 3 min after the ablation process. Corneal thinning due to evaporation was studied on a separate patient with the OLCR instrument, and it was found to be ?0.14 µm/s during the first 5 min after epithelium removal. This baseline corneal thinning rate was used as a fit parameter to calculate actual from measured ablation depths. The measurements showed a maximum difference of ±10 µm between planned ablations (34–92 µm) and measured ablation depths.

Parametric and nonparametric data processing schemes for analyzing translating laser speckle data used to investigate the mechanical behavior of biological tissues are examined. Crosscorrelation, minimum mean square estimator, maximum likelihood, and maximum entropy approaches are discussed and compared on speckle data derived from cortical bone samples undergoing dynamic loading. While it was not the purpose of this paper to demonstrate that one processing technique is superior to another, maximum likelihood and maximum entropy approaches are shown to be particularly useful when the observed speckle motion is small.

Solid protein solder-doped polymer membranes were developed for laser-assisted tissue repair. Biodegradable polymer membranes of controlled porosity were fabricated with poly(L-lactic-coglycolic acid) (PLGA), poly(ethylene glycol) (PEG), and salt particles, using a solvent-casting and particulate-leaching technique. The membranes provided a porous scaffold that readily absorbed the traditional protein solder composed of serum albumin, indocyanine green dye, and de-ionized water. In vitro investigations were conducted to assess the influence of various processing parameters on the strength of tissue repairs formed using the new membranes. These parameters included PLGA copolymer and PLGA/PEG blend ratios, membrane pore size, initial albumin weight fraction, and laser irradiance used to denature the solder. Altering the PLGA copolymer ratio had little effect on repair strength, however such variations are known to influence the degradation rate of the membranes. The repair strength increased with increased membrane pore size and bovine serum albumin concentration. The addition of PEG during the membrane casting stage increased the flexibility of the membranes but not necessarily the repair strength. Typically, the repair strength increased with increasing irradiance from 12 to 18 W/cm2. The new solder-doped polymer membranes provided all of the benefits associated with solid protein solders including high repair strength and improved edge coaptation. In addition, the flexible, moldable nature of the new membranes offers the capability of tailoring the membranes to a wide range of clinically relevant geometries.

In vivo imaging of cells tagged with light-emitting probes, such as firefly luciferase or fluorescent proteins, is a powerful technology that enables a wide range of biological studies in small research animals. Reporters with emission in the red to infrared (>600 nm) are preferred due to the low absorption in tissue at these wavelengths. Modeling of photon diffusion through tissue indicates that bioluminescent cell counts as low as a few hundred can be detected subcutaneously, while ~106 cells are required to detect signals at ~2 cm depth in tissue. Signal-to-noise estimates show that cooled back-thinned integrating charge coupled devices (CCDs) are preferred to imageintensified CCDs for this application, mainly due to their high quantum efficiency (~85%) at wavelengths >600 nm where tissue absorption is low. Instrumentation for in vivo imaging developed at Xenogen is described and several examples of images of mice with bioluminescent cells are presented.

Simple analytical expressions for the point spread function (PSF) at different depths can save computation time and improve the performance of inverse algorithms for optical imaging. In particular, application of such formulas simplifies quantification of the optical characteristics of tissue abnormalities inside highly scattering media. Earlier it was shown within the random walk theory framework that the PSF for time-resolved transillumination imaging through a highly scattering slab is well represented by a Gaussian. We have experimentally validated a simple formula of the random walk model for the effective width of this Gaussian, as a function of time delay, at different depths of the target. Presented analysis of published experimental data, concerning effective width of the PSF, for a slab of considerably smaller thickness also demonstrates good agreement between the data and predictions of our model. This PSF width determines spatial resolution of the time-resolved imaging and is widely discussed in the literature.

Ultrashort infrared laser pulses were transmitted through excised female breast tissue. The resulted signal was recorded by a streak camera with a time resolution of the order of a few ps. Experimental data of the temporal spread of the ultrashort pulse during the transmission through the tissue have been analyzed using the Patterson analytical expression derived from the diffusion theory. This resulted in the calculation of the absorption and reduced scattering coefficients, which are related to the optical characteristics of each type of tissue. The goal of the study was to use the theoretical values of the coefficients to discriminate different kinds of tissue.

We report a novel support, concomitant attachment chemistry, and a fluorescent imaging system for DNA microarrays. The support consists of soda lime glass coated with a layer of chromium, which eliminates any autofluorescence from the underlying glass substrate and reduces nonspecific probe binding. Attachment of DNA fragments exceeding 300 nucleotides in length is achieved without chemical modifications of either the chrome surface or the DNA itself. The charge coupled device (CCD)-based imaging system employs a 175 W xenon arc lamp as the light source, allowing the use of many different fluorophors. A 14mmx9 mm sample area is imaged with a single exposure, which takes between 5 and 20 s for each color plane in typical genomic comparative genomic hybridization type assays. The spatial resolution is limited only by the pixel size of the CCD chip (9 µm). The oblique illumination geometry combined with effective background reduction afforded by the chromium surface enables the system to achieve a detection limit of ,5x107 fluorophors/cm2 with 10 s integration. In a model system with arrayed lambda DNA targets a dose response was observed over four orders of magnitude in response to hybridizations with increasing amounts of the fluorescent labeled lambda probe.

A simple method to evaluate the hemoglobin oxygen saturation and relative hemoglobin concentration in a tissue from diffuse reflectance spectra in the visible wavelength range is put forward in this paper. It was assumed that while oxygenated and deoxygenated hemoglobin contributions to light attenuation are strongly variable functions of wavelength, all other contributions to the attenuation including scattering are smooth wavelength functions and can be approximated by Taylor series expansion. Based on this assumption, a simple, robust algorithm suitable for real time monitoring of the hemoglobin oxygen saturation in the tissue has been derived. This algorithm can be used with different fiber probe configurations for delivering and collecting light passed through the tissue. An experimental technique using this algorithm has been developed for in vivo monitoring during artery occlusion and in vitro monitoring of blood samples. The experimental results obtained are presented in the paper.

Diffusion theory is often used to model the transport of light within tissue. It can be used to calculate the light fluence rate in tissue, for example, during photodynamic therapy, or to measure the absorption and scattering properties of tissue. For both of these applications, the influence of the interface between the tissue and the exterior medium on the fluence rate inside the tissue must be known in order to make accurate calculations. We present an experimental investigation of the effect of the refractive index mismatch at the tissue interface on the internal light fluence rate and on the spatially resolved diffuse reflectance as the boundary conditions of the tissue/ external medium are changed. The effects of changing the relative refractive index at the boundary are compared to predictions of diffusion theory. The effect of the refractive index mismatch is predicted correctly by diffusion theory.

We report the first application of high-speed fiber-based polarization sensitive optical coherence tomography (PS-OCT) to image burned tissue in vivo. Thermal injury denatures collagen in skin and PS-OCT can measure the reduction in collagen birefringence using depth resolved changes in the polarization state of light propagated in, and reflected from, the tissue. Stokes vectors were calculated for each point in a scan and birefringence relative to incident polarization determined using four incident polarization states. Using a high-speed fiber-based PS-OCT system on rat skin burned for varying periods of time, a correlation between birefringence and actual burn depth determined by histological analysis was established. In conclusion, PSOCT has potential use for noninvasive assessment of burn depth.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope’s effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.