Imaging human epithelial properties with polarized light-scattering spectroscopy.

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.

Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the potential of 3 spectroscopic techniques (fluorescence, reflectance, and light-scattering spectroscopy) individually and in combination, for evaluating low- and high-grade dysplasia in patients with Barrett's esophagus (BE). METHODS: Fluorescence spectra at 11 excitation wavelengths and a reflectance spectrum were acquired in approximately 1 second from each site before biopsy using an optical fiber probe. The measured fluorescence spectra were combined with the reflectance spectra to extract the intrinsic tissue fluorescence. The reflectance spectra provided morphologic information about the bulk tissue, whereas light-scattering spectroscopy was used to determine cell nuclear crowding and enlargement in Barrett's epithelium. RESULTS: Significant differences were observed between dysplastic and nondysplastic BE in terms of intrinsic fluorescence, bulk scattering properties, and levels of epithelial cell nuclear crowding and enlargement. The combination of all 3 techniques resulted in superior sensitivity and specificity for separating high-grade from non-high-grade and dysplastic from nondysplastic epithelium. CONCLUSIONS: Intrinsic fluorescence, reflectance, and light-scattering spectroscopies provide complementary information about biochemical and morphologic changes that occur during the development of dysplasia. The combination of these techniques (Tri-Modal Spectroscopy) can serve as an excellent tool for the evaluation of dysplasia in BE.

Surface-enhanced and normal stokes and anti-stokes Raman spectroscopy of single-walled carbon nanotubes.

Surface enhancement factors of at least 10(12) for the Raman scattering of single-walled carbon nanotubes in contact with fractal silver colloidal clusters result in measuring very narrow Raman bands corresponding to the homogeneous linewidth of the tangential C-C stretching mode in semiconducting nanotubes. Normal and surface-enhanced Stokes and anti-Stokes Raman spectra are discussed in the framework of selective resonant Raman contributions of semiconducting or metallic nanotubes to the Stokes or anti-Stokes spectra, respectively, of the population of vibrational levels due to the extremely strong surface-enhanced Raman process, and of phonon-phonon interactions.

Endoscopic detection of dysplasia in patients with Barrett's esophagus using light-scattering spectroscopy.

BACKGROUND & AIMS: We conducted a study to assess the potential of light-scattering spectroscopy (LSS), which can measure epithelial nuclear enlargement and crowding, for in situ detection of dysplasia in patients with Barrett's esophagus. METHODS: Consecutive patients with suspected Barrett's esophagus underwent endoscopy and systematic biopsy. Before biopsy, each site was sampled by LSS using a fiberoptic probe. Diffusely reflected white light was spectrally analyzed to obtain the size distribution of cell nuclei in the mucosal layer, from which the percentage of enlarged nuclei and the degree of crowding were determined. Dysplasia was assigned if more than 30% of the nuclei exceeded 10 microm and the histologic findings compared with those of 4 pathologists blinded to the light-scattering assessment. The data were then retrospectively analyzed to further explore the diagnostic potential of LSS. RESULTS: Seventy-six sites from 13 patients were sampled. All abnormal sites and a random sample of nondysplastic sites were reviewed by the pathologists. The average diagnoses were 4 sites from 4 different patients as high-grade dysplasia (HGD), 8 sites from 5 different patients as low-grade dysplasia (LGD), 12 as indefinite for dysplasia, and 52 as nondysplastic Barrett's. The sensitivity and specificity of LSS for detecting dysplasia (either LGD or HGD) were 90% and 90%, respectively, with all HGD and 87% of LGD sites correctly classified. Decision algorithms using both nuclear enlargement and crowding further improved diagnostic accuracy, and accurately classified samples into the 4 histologic categories. CONCLUSIONS: LSS can reliably detect LGD and HGD in patients with Barrett's esophagus.

Feasibility of field-based light scattering spectroscopy.

Light scattering spectroscopy (LSS) is a new technique capable of accurately measuring the features of nuclei and other cellular organelles in situ. We present the considerations required to implement and interpret field-based detection in LSS, where the scattered electric field is detected interferometrically, and demonstrate that the technique is experimentally feasible. A theoretical formalism for modeling field-based LSS signals based on Mie scattering is presented. Phase-front uniformity is shown to play an important and novel role. Results of heterodyne experiments with polystyrene microspheres that localize LSS signals to a region about 30 microns in axial extent are reported. In addition, differences between field-based LSS and the earlier intensity-based LSS are discussed.

Optical computed tomography in a turbid medium using early arriving photons.

We employ photon migration to image absorbing objects embedded in a turbid medium. For improved resolution, we use early arriving photons (a few hundred picoseconds in excess of the time of flight), a regime in which the diffusion approximation breaks down. Our image reconstruction method is based on extension of x-ray computed tomography (CT) to the optical regime. The CT algorithm must be generalized to take into account the distributions of photon paths. We express the point spread function (PSF) in terms of the Green's function for the transport equation. This PSF then provides weighting functions for use in a generalized series expansion method of x-ray CT. Experiments were performed on a turbid medium with scattering and absorption properties similar to those of human breast tissue. Multiple absorbers were embedded into the medium to mimic tumors. Coaxial transmission scans were collected in two projections, and the early-time portions were analyzed. Through accurate modeling, we could remove the blurring associated with multiple scattering and obtain high-resolution images. Our results show that the diffusion approximation PSF is inadequate to describe the early arriving photons. A PSF incorporating causality is required to reconstruct accurate images of turbid media.

Physical properties of hydrated tissue determined by surface interferometry of laser-induced thermoelastic deformation.

Knee meniscus is a hydrated tissue; it is a fibrocartilage of the knee joint composed primarily of water. We present results of interferometric surface monitoring by which we measure physical properties of human knee meniscal cartilage. The physical response of biological tissue to a short laser pulse is primarily thermomechanical. When the pulse is shorter than characteristic times (thermal diffusion time and acoustic relaxation time) stresses build and propagate as acoustic waves in the tissue. The tissue responds to the laser-induced stress by thermoelastic expansion. Solving the thermoelastic wave equation numerically predicts the correct laser-induced expansion. By comparing theory with experimental data, we can obtain the longitudinal speed of sound, the effective optical penetration depth and the Grüneisen coefficient. This study yields information about the laser tissue interaction and determines properties of the meniscus samples that could be used as diagnostic parameters.

Spatial coherence of forward-scattered light in a turbid medium.

We study spatially coherent forward-scattered light propagating in a turbid medium of moderate optical depth (0-9 mean free paths). Coherent detection was achieved by using a tilted heterodyne geometry, which desensitizes coherent detection of the attenuated incident light. We show that the degree of spatial coherence is significantly higher for light scattered only once in comparison with that for multiply scattered light and that it approaches a small constant value for large numbers of scattering events.

Diffuse reflectance spectroscopy of human adenomatous colon polyps in vivo.

Diffuse reflectance spectra were collected from adenomatous colon polyps (cancer precursors) and normal colonic mucosa of patients undergoing colonoscopy. We analyzed the data by using an analytical light diffusion model, which was tested and validated on a physical tissue model composed of polystyrene beads and hemoglobin. Four parameters were obtained: hemoglobin concentration, hemoglobin oxygen saturation, effective scatterer density, and effective scatterer size. Normal and adenomatous tissue sites exhibited differences in hemoglobin concentration and, on average, in effective scatterer size, which were in general agreement with other studies that employ standard methods. These results suggest that diffuse reflectance can be used to obtain tissue information about tissue structure and composition in vivo.

Raman spectroscopy and fluorescence photon migration for breast cancer diagnosis and imaging.

We are developing optical methods based on near infrared Raman spectroscopy and fluorescence photon migration for diagnosis and localization of breast cancer. We demonstrate the ability of Raman spectroscopy to classify accurately normal, benign and malignant breast tissues, an important step in developing Raman spectroscopic needle probes as a tool for improving the accuracy of needle biopsy. We also show that photon migration imaging can be used to localize accurately small fluorescent objects imbedded in a thick turbid medium with realistic optical properties, thus demonstrating the potential of this technique for optical imaging.

Distribution of the paths of early-arriving photons traversing a turbid medium.

We describe experiments to measure the spatial and the temporal distribution of photons traversing a turbid medium in the early-arriving regime in which the photons are multiply scattered but are not completely randomized. The photon paths are resolved temporally by a streak camera and spatially by an adjustable absorbing screen with a small aperture. The results are compared with predictions of a theory based on path integrals (PIs) and with the standard diffusion approximation. The PI theory agrees with the data for both long and short times of flight; this agreement is in contrast to the diffusion approximation, which fails for short times. An alternative PI calculation, based on the use of an effective Lagrangian, also agrees with the experiments. PI theory succeeds because it preserves causality. The implications for optical tomography are discussed.

Fluorescence tomographic imaging in turbid media using early-arriving photons and Laplace transforms.

We present a multichannel tomographic technique to detect fluorescent objects embedded in thick (6.4 cm) tissue-like turbid media using early-arriving photons. The experiments use picosecond laser pulses and a streak camera with single photon counting capability to provide short time resolution and high signal-to-noise ratio. The tomographic algorithm is based on the Laplace transform of an analytical diffusion approximation of the photon migration process and provides excellent agreement between the actual positions of the fluorescent objects and the experimental estimates. Submillimeter localization accuracy and 4- to 5-mm resolution are demonstrated. Moreover, objects can be accurately localized when fluorescence background is present. The results show the feasibility of using early-arriving photons to image fluorescent objects embedded in a turbid medium and its potential in clinical applications such as breast tumor detection.

Photon migration of near-diffusive photons in turbid media: a Lagrangian-based approach.

We show that light transport in a turbid medium can be described by a path integral with a quasi-particle Lagrangian. The most probable trajectory over which photons can be found can be obtained from this Lagrangian. This approach extends the diffusion approximation to the near-diffusive regime, in which photons travel only a few transport mean free paths.

The thermoelastic basis of short pulsed laser ablation of biological tissue.

Strong evidence that short-pulse laser ablation of biological tissues is a photomechanical process is presented. A full three-dimensional, time-dependent solution to the thermoelastic wave equation is compared to the results of experiments using an interferometric surface monitor to measure thermoelastic expansion. Agreement is excellent for calibrations performed on glass and on acrylic at low laser fluences. For cortical bone, the measurements agree well with the theoretical predictions once optical scattering is included. The theory predicts the presence of the tensile stresses necessary to rupture the tissue during photomechanical ablation. The technique is also used to monitor the ablation event both before and after material is ejected.

Mechanisms of meniscal tissue ablation by short pulse laser irradiation.

A new experimental technique was developed to study short-pulsed laser ablation of biologic tissues (human meniscus and bovine tibial bone), water, and acrylic. The experimental technique was based on interferometric monitoring of the motion of the tissue surface to measure its laser-induced expansion after irradiation. The thermoelastic expansion of these materials after laser irradiation under subablation threshold was examined to determine its role in the initiation of ablation. The experimentally observed surface expansion of cortical bone and acrylic was in agreement with theoretical predictions. The movement of meniscal tissue was similar to that shown by water. The latter 2 materials showed additional features consistent with the growth and collapse of cavitation bubbles. The exact role of cavitation in the irradiation of meniscal tissue by laser light remains unknown, but may represent a clinically important mode of tissue ablation and postirradiation trauma.

Time-resolved multichannel imaging of fluorescent objects embedded in turbid media.

We present a multichannel detection technique for three-dimensional imaging of objects embedded in turbid media by using time-resolved fluorescence. By using a streak camera, we can obtain the experimental data in a single measurement. The data, analyzed by means of a triangulation algorithm, provide accurate localization of a fluorescent object for path lengths of up to 120 scattering mean free paths. The results demonstrate the feasibility of combining fluorescence spectroscopy with time-resolved optical tomography for localizing and identifying embedded objects.

Three-dimensional imaging of objects embedded in turbid media with fluorescence and Raman spectroscopy.

We present a single-ended technique for three-dimensional imaging of objects embedded in a turbid medium by the use of time-resolved fluorescence emission or Raman scattering. The technique uses the earliest arriving photons, which we show are not sensitive to the relatively long fluorescence lifetime, and thus can be used to extract the desired spatial information accurately, even at a distance equivalent to 100 mean free paths. The results also demonstrate the feasibility and the potential of one's combining time-resolved optical tomography with fluorescence or Raman spectroscopy to localize and identify the embedded objects. This technique may be valuable for the diagnosis of disease in highly scattering human tissue because it can provide spatial and biochemical information about the composition of embedded lesions.

Laser-induced thermoelastic deformation: a three-dimensional solution and its application to the ablation of biological tissue.

Under certain conditions, laser light incident on a target material can induce an explosive removal of some material, a process called laser ablation. The photomechanical model of laser ablation asserts that this process is initiated when the laser-induced stresses exceed the strength of the material in question. Although one-dimensional calculations have shown that short pulsed lasers can create significant transient tensile stresses in target materials, the stresses last for only a few nanoseconds and the spatial location of the peak stresses is not consistent with experimental observations of material failure in biological tissues. Using the theory of elasticity, analytical expressions have been derived for the thermoelastic stresses and deformations in an axially symmetric three-dimensional solid body caused by the absorption of laser light. The full three-dimensional solution includes three stresses, radial, circumferential and shear, which are necessarily absent in the simple one-dimensional solution. These stresses have long-lived components that exist for eight orders of magnitude longer in time than the acoustic transients, an important point when the details of dynamic fracture are considered. Many important qualitative features are revealed including the spatial location of the peak stresses, which is more consistent with experimental observations of failure.

Interferometric surface monitoring of biological tissue to study inertially confined ablation.

We present results from the application of laser interferometry to the study of short-pulsed laser ablation of biological tissue. The mechanical response of tissue to laser-induced stress is examined under subthreshold conditions to determine its role in initiating the ablation process. A theoretical model is developed to relate this surface displacement to the pressure within the tissue and the mechanical properties of the tissue. In the experiment, a 7.5 ns pulse of 355 nm light was used to irradiate bovine shank bone, human meniscus, and an aqueous dye solution. Interferometric monitoring of the tissue surface was used to determine its motion after laser irradiation. The surface movement of bone was qualitatively consistent with the theoretical predictions of the model. The movement of meniscus and an aqueous dye solution showed additional features that are consistent with the growth and collapse of cavitation bubbles.