Comparison of elastic scattering spectroscopy with histology in ex vivo prostate glands: potential application for optically guided biopsy and directed treatment.

The false-negative rate of ultrasound-guided sextant prostate biopsy has been estimated to be as high as 35 %. A significant percentage (10-35 %) of these prostate cancers diagnosed at a second or later attempt are high grade and, therefore, potentially lethal. We discuss the feasibility for performing optically guided biopsy using elastic scattering spectroscopy (ESS) to reduce sampling errors and improve sensitivity. ESS measurements were performed on 42 prostate glands ex vivo and correlated with standard histopathological assessment. Sliced glands were examined with wavelength ranges of 330-760 nm. The ESS portable system used a new fiber-optic probe with integrated cutting tool, designed specifically for ex vivo pathology applications. ESS spectra were grouped by diagnosis from standard histopathological procedure and then classified using linear support vector machine. Preliminary data are encouraging. ESS data showed strong spectral trends correlating with the histopathological assignments. The classification results showed a sensitivity of 0.83 and specificity of 0.87 for distinguishing dysplastic prostatic tissue from benign prostatic tissue. Similar results were obtained for distinguishing dysplastic prostatic tissue from prostatitis with a sensitivity and specificity of 0.80 and 0.88, respectively. The negative predictive values obtained with ESS are better than those obtained with transrectal ultrasound (TRUS)-guided core-needle biopsy.

Optical scanning for rapid intraoperative diagnosis of sentinel node metastases in breast cancer.

BACKGROUND: Intraoperative diagnosis of sentinel node metastases enables an immediate decision to proceed to axillary lymph node dissection, avoiding a second operation in node-positive women with breast cancer. METHODS: An optical scanner was developed that interrogated the cut surface of bivalved, but otherwise unprocessed, sentinel lymph nodes with pulses of white light by elastic scattering spectroscopy (ESS). The scattered light underwent spectral analysis, and individual spectra were initially correlated with conventional histology to develop a diagnostic algorithm. This algorithm was used to create false colour-coded maps of scans from an independent set of nodes, and the optimal criteria for discriminating between normal and cancer spectra were defined statistically. RESULTS: The discriminant algorithm was developed from a training set of 2989 spectra obtained from 30 metastatic and 331 normal nodes. Subsequent scans from 129 independent nodes were analysed. The scanner detected macrometastases (larger than 2 mm) with a sensitivity of 76 per cent (69 per cent including micrometastases) and specificity of 96 per cent. CONCLUSION: In this proof-of-principle study, the ESS results were comparable with current intraoperative diagnostic techniques of lymph node assessment.

Assessment of oral premalignancy using elastic scattering spectroscopy.

Optical spectroscopy systems have been involved in various clinical fields; however the main interest is still in the diagnosis of premalignant/malignant lesions. The aim of this study was to compare findings of Elastic Scattering Spectroscopy (ESS) with histopathology of oral tissues to see if this technique could be used as an adjunct or alternative to histopathology in identifying dysplasia. The technique involves the use of Mie scattering and is a simple non-invasive method of tissue interrogation. Twenty-five oral sites from 25 patients who presented with oral leukoplakia were examined by ESS using a pulsed xenon-arc lamp. Surgical biopsies were acquired from each of the examination sites. The results of the acquired spectra were then compared with histopathology. Two sets of spectra were obtained, and by using a linear discriminant analysis, a sensitivity of 72% and a specificity of 75% were obtained. These results are promising and could suggest that ESS may be able to identify dysplasia in oral tissues. To prove the usefulness of the ESS in dysplasia detection in oral tissues conclusively, a larger body of data is needed. We aim to continue this study to obtain more data in an attempt to increase the accuracy of the technique. Large, multi-centre trails are needed for each anatomical site, in order to gather more information about the differences between normal and dysplastic tissue.

Diagnosis of breast cancer using elastic-scattering spectroscopy: preliminary clinical results.

We report on the first stages of a clinical study designed to test elastic-scattering spectroscopy, mediated by fiberoptic probes, for three specific clinical applications in breast-tissue diagnosis: (1) a transdermal-needle (interstitial) measurement for instant diagnosis with minimal invasiveness similar to fine-needle aspiration but with sensitivity to a larger tissue volume, (2) a hand-held diagnostic probe for use in assessing tumor/resection margins during open surgery, and (3) use of the same probe for real-time assessment of the "sentinel" node during surgery to determine the presence or absence of tumor (metastatic). Preliminary results from in vivo measurements on 31 women are encouraging. Optical spectra were measured on 72 histology sites in breast tissue, and 54 histology sites in sentinel nodes. Two different artificial intelligence methods of spectral classification were studied. Artificial neural networks yielded sensitivities of 69% and 58%, and specificities of 85% and 93%, for breast tissue and sentinel nodes, respectively. Hierarchical cluster analysis yielded sensitivities of 67% and 91%, and specificities of 79% and 77%, for breast tissue and sentinel nodes, respectively. These values are expected to improve as the data sets continue to grow and more sophisticated data preprocessing is employed. The study will enroll up to 400 patients over the next two years.

Non-invasive measurement of chemotherapy drug concentrations in tissue: preliminary demonstrations of in vivo measurements.

Measurements of the tissue concentrations of two chemotherapy agents have been made in vivo on an animal tumour model. The method used is based on elastic scattering spectroscopy (ESS) and utilizes a fibre-optic probe spectroscopic system. A broadband light source is used to acquire data over a broad range of wavelengths and, therefore, to facilitate the separation of absorptions from various chromophores. The results of the work include measurements of the time course of the drug concentrations as well as a comparison of the optical measurements with high performance liquid chromatography (HPLC) analysis of the drug concentrations at the time of sacrifice. It is found that the optical measurements correlate linearly with HPLC measurements, but give lower absolute values.

Noninvasive, in-situ measurement of drug concentrations in tissue using optical spectroscopy.

A small, lightweight system capable of noninvasive measurement of drug concentrations in tissue, without the use of reagents, would be advantageous for the study of the drug pharmacokinetics in space-travel applications. We have applied elastic-scattering spectroscopy (ESS) for noninvasive, real-time in vivo measurement of the concentrations of certain drugs in tissue, utilizing a simple fiber-optic-probe spectroscopic system. The system uses a broadband light source, enabling the detection of compounds with absorption bands in most regions of the visible, and potentially in the near-infrared (NIR) to 1700 nm. Subcutaneous tumors were grown in 4 Nude mice; the mice were treated with one of two chemotherapy agents, and the ESS system was used to perform pharmacokinetic measurements on the tumors following drug administration. Time histories of the drug concentrations in the tumors agreed with the known pharmacokinetics of the two drugs, and HPLC assays following sacrifice showed good relative correlation with the ESS values. Most photodynamic therapy agents and many chemotherapy drugs, including some that are not fluorescent, are ideal candidates for the ESS system. Importantly, the method can provide absolute measurements of compound concentrations.

Measuring absorption coefficients in small volumes of highly scattering media: source-detector separations for which path lengths do not depend on scattering properties.

The noninvasive measurement of variations in absorption that are due to changes in concentrations of biochemically relevant compounds in tissue is important in many clinical settings. One problem with such measurements is that the path length traveled by the collected light through the tissue depends on the scattering properties of the tissue. We demonstrate, using both Monte Carlo simulations and experimental measurements, that for an appropriate separation between light-delivery and light-collection fibers the path length of the collected photons does not depend on scattering parameters for the range of parameters typically found in tissue. This is important for developing rapid, noninvasive, and inexpensive methods for measuring absorption changes in tissue.

Ultraviolet and visible spectroscopies for tissue diagnostics: fluorescence spectroscopy and elastic-scattering spectroscopy.

We review the application of fluorescence spectroscopy and elastic-scattering spectroscopy, over the ultraviolet-to-visible wavelength range, to minimally invasive medical diagnostics. The promises and hopes, as well as the difficulties, of these developing techniques are discussed.

Predictions and measurements of scattering and absorption over broad wavelength ranges in tissue phantoms.

Predictions from Mie theory regarding the wavelength dependence of scattering in tissue from the near UV to the near IR are discussed and compared with experiments on tissue phantoms. For large fiber separations it is shown that rapid, simultaneous measurements of the elastic scatter signal for several fiber separations can yield the absorption coefficient and reduced scattering coefficient. With this information, the size of the scattering particles can be estimated, and this is done for Intralipid. Measurements made at smaller source detector separations support Mie theory calculations, demonstrating that the sensitivity of elastic scatter measurements to morphological features, such as scatterer size, is enhanced when the distance between the source and detector fibers is small.

Influence of particle size and concentration on the diffuse backscattering of polarized light from tissue phantoms and biological cell suspensions.

We present experimental results that show the spatial variations of the diffuse-backscattered intensity when linearly polarized light is incident upon highly scattering media. Experiments on polystyrene-sphere and Intralipid suspensions demonstrate that the radial and azimuthal variations of the observed pattern depend on the concentration, size, and anisotropy factor g of the particles that constitute the scattering medium. Measurements performed on biological-cell suspensions show the potential of this method for cell characterization.

Influence of the scattering phase function on light transport measurements in turbid media performed with small source-detector separations.

Many methods of optical tissue diagnosis require that measurements be performed with small source-detector separations in a backscatter geometry. Monte Carlo simulations are used to demonstrate that for these situations light transport depends on the exact form of the angular scattering probability distribution, P(theta). Simulations performed with different forms of P(theta) with the same value of ?cos theta? result in the collection of significantly different fractions of the incident photons, particularly when small-numerical-aperture delivery and collection fibers are employed. More photons are collected for the distribution that has a higher probability of scattering events with theta > 125 degrees . For the clinically relevant optical parameters employed here, the differences in light collection are >60%.

Spectroscopic diagnosis of bladder cancer with elastic light scattering.

BACKGROUND AND OBJECTIVES: Recently, significant progress has been made toward the development of optical, noninvasive medical diagnostics. The goal of this study was to evaluate elastic scatter measurements as a tool for diagnosing bladder cancer. STUDY DESIGN/MATERIALS AND METHODS: In vivo measurements on 10 patients with suspected bladder cancer were made with the optical biopsy system (OBS) developed at Los Alamos National Laboratory. Elastic-scatter spectra over the wavelength range 250-800 nm were obtained using a fiber-optic probe through one of the lumens of a urological cystoscope. Measurements were made on putatively normal areas and areas of uncertain abnormality, as well as those suspected to be cancerous. After measurements were made with the OBS, biopsy samples were taken at the measurement sites. Comparisons of the histopathology and the optical spectra were then made. RESULTS AND CONCLUSIONS: A diagnostic algorithm for distinguishing malignant from nonmalignant tissue based on the values of the slopes over the wavelength range 330-370 nm has a sensitivity of 100% and specificity of 97% for the limited number of patients in this study.

Microwave absorption spectroscopy of DNA.

By utilizing a novel approach to microwave spectrometry, we have measured the absolute absorption spectrum of plasmid DNA (pUC8.c2), in buffered aqueous solution, from 5 to 20 GHz. Our technique does not suffer from the same experimental difficulties that plague other methods. We observe no absorption resonances in this frequency range, but we do see broadband differences, between DNA and pure buffer, that are attributable to changes in the ionic conductivity of the solutions. These results constitute the first verification, by a totally different technique, of the absence of resonances in the microwave absorption spectrum of DNA, and the first data obtained by any technique in the 10-20-GHz band.

Transient fluorescence in synchronously dividing Escherichia coli.

Using a spectrometer equipped with an optical multichannel analyzer as the detector, we observed the Stokes laser-Raman spectra of metabolically synchronous Escherichia coli from 100 to 2100 cm-1. After more than 400 separate recordings, at cell concentrations of 10(7)-10(8) per ml, no Raman lines attributable to the metabolic process nor to the cells themselves were found. However, we did find that synchronous E. coli cultures become more fluorescent during a limited phase of the division cycle. This transient increase in fluorescence may be ascribed to a variation in the redox state of a chemical species within the bacteria or to a variation of the intracellular optical field. The effect is reproducible in synchronous cultures and it is not seen in asynchronous ones. The results suggest that spectral features seen in previous laser-Raman spectra of synchronous bacteria (taken with scanning monochromators) are due to a time-dependent variation in bacterial fluorescence.

Quenching of backward stimulated Raman scattering by broadband forward Raman radiation.

Experiments on backward stimulated Raman scattering of an injection-locked XeF (351.1-nm) laser beam are presented. Because the XeF laser is inhomogeneously broadened, its output is composed of both a narrow-band component (50-60% of the energy) and an unlocked broadband component. It is found that the presence of forward Raman scattering from the broadband unlocked component of the XeF energy terminates the backward Raman scattering of the narrow- (~1-GHz) bandwidth-locked component of the radiation. The results are of a general nature and would apply to scattering from any inhomogeneously broadened injection-locked laser.

Transform-limited-bandwidth injection locking of an XeF laser with an Ar-ion laser at 3511 A.

We report attainment of transform-limited bandwidth in an XeF laser by injection locking a positive-branch confocal unstable resonator with 1 W of power from an Ar-ion laser at 3511 A. A diffraction-limited beam of >2-MW power, with a bandwidth of ~50 MHz (~1 x 10(-4) A), is generated. We find that since the XeF UV emission lines start from differentupper vibrational levels of the B state, ~40% of the B ? X energy is not available to be channeled into the locked line.

Efficient phase conjugation of an ultraviolet XeF laser beam by stimulated Brillouin scattering.

We report efficient, diffraction-limited, phase conjugation of an XeF (3511-A) laser beam using stimulated Brillouin scattering. Approximately 70% of the 1-GHz bandwidth-locked portion of an injection-locked XeF laser output is phase conjugated by focusing the laser beam at 5 GW/cm (2) into hexane or isopropanol.

Attainment of the theoretical minimum input power for injection locking of an unstable-resonator KrF laser.

We report attaining a ratio of output to injected power as high as 10(8) in a KrF system, the limit being reached when injected power equals the noise per mode in the resonator. We find that, because of coherence effects, adjustment of the cavity length for correct value of the equivalent Fresnel number can change the threshold power for frequency locking by at least 2 orders of magnitude. The laser's locked bandwidth is <0.1 A, and the line can be tuned over a range exceeding 10 A. The beam divergence is near (i.e., 1.5x) the diffraction limit.

Beam-diagnostics techniques for multiterawatt CO(2) lasers.

The requirements of the Laser Fusion Program place severe demands on the ability to monitor the important parameters of the laser beams. This has resulted in the development of novel and imaginative methods to provide the various required measurements. The design and implementation of a beam-diagnostics system for the Helios laser fusion facility at Los Alamos are described.