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.

In vivo spectrophotometric evaluation of normal, lesional, and laser-treated skin in patients with port-wine stains.

The influence of patient age and argon laser therapy on port-wine stains (PWS) was studied quantitatively in 16 patients aged 15-64 years using a spectrophotometer and computer graphics/statistics program. Normalized reflectance curves revealed a 10-20% decrease with age in the reflectance of normal skin from 400 nm to 650 nm, with an even more pronounced reflectance decrease in the region of peak deoxyhemoglobin absorption at approximately 555 nm. In each patient, PWS reflectance was less than that in the normal skin, as expected, and the average discrepancy increased with age from approximately 25% to 50%, with further reduction at 555 nm. The data suggest that with advancing age, both normal skin and PWS have a greater total hemoglobin content and an increased proportion of deoxyhemoglobin, consistent with increasing vascular dilation and tortuosity; and that the age-associated changes in PWS are an exaggeration of those in normal skin. Laser-treated PWS in both young and old patients had reflectance curves indistinguishable from those of untreated PWS in young patients. This implies, contrary to published clinical impressions, that in the absence of scarring the results of argon laser therapy are the same in young and old patients, but that only older patients experience a significant color shift in the lesion.

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.

Reagentless blood analysis by near-infrared Raman spectroscopy.

BACKGROUND: Raman spectroscopy has advantages over infrared absorption spectroscopy. Combined with a novel multivariate technique, hybrid linear analysis (HLA), low prediction error is expected. METHODS: A near-infrared (NIR) light source excited Raman signals, and a charge coupled device (CCD) camera was used to collect the signal. Samples were collected from 69 individuals for 7 weeks. The standard multivariate calibration technique, partial least squares (PLS) and HLA were both used to analyze the collected spectra. A Clarke error grid was used to evaluate the usefulness of the glucose measurement in serum. RESULTS: The root mean squared error of prediction (RMSEP) for glucose in serum obtained with PLS is 21 mg/dL, and the RMSEP obtained with HLA is 17 mg/dL. In whole blood, the PLS RMSEP for glucose was 79 mg/dL, and HLA predictions had an RMSEP of 63 mg/dL. CONCLUSIONS: The measurement technique was robust over the 7-week period. HLA was shown to generate a lower prediction error than PLS. The predictions by both PLS and HLA were clinically acceptable. The result with whole blood requires further improvement.

Near-infrared fluorescence spectroscopy detects Alzheimer's disease in vitro.

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.

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.

Prospects for in vivo Raman spectroscopy.

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

Diagnosis of head and neck precancerous lesions in an animal model using fluorescence spectroscopy.

Laser-induced fluorescence (LIF) of tissues depends on their biochemical and histomorphologic characteristics. LIF spectroscopic properties of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced precancerous and early cancerous lesions in a hamster buccal pouch mucosa model were studied. Fluorescence spectra from neoplastic lesions showed a characteristic fluorescence peak in the red region of the visible spectrum centered between 630 and 640 nm when excited with 410-nm light. Using this as a diagnostic criterion, 45 of 49 lesions studied were correctly diagnosed, including early dysplastic lesions. Follow-up study of four dysplastic lesions over 2 weeks revealed an increase in red fluorescence intensity. The findings of these experiments suggest that LIF spectroscopy may be a valuable noninvasive technique not only for early diagnosis of head and neck cancer, but also to probe a possible biochemical surrogate biomarker in the follow-up of suspected lesions.

Early diagnosis of upper aerodigestive tract cancer by autofluorescence.

OBJECTIVE: To explore the potential of autofluorescence spectroscopy as a tool for early detection of upper aerodigestive tract cancer. DESIGN: Autofluorescence spectral characteristics of 19 untreated oral and oropharyngeal lesions in 13 patients were studied with excitation wavelengths of 370 and 410 nm generated by a nitrogen pumped dye laser. Ten healthy volunteers were recruited to characterize the fluorescence spectra of normal mucosa at different oral sites and to study individual variations. Fluorescence intensity and line shape of the spectra from lesions were compared with the same parameters from the contralateral control site in the same individual. SETTING: Otolaryngology Research Center, Department of Otolaryngology-Head and Neck Surgery, New England Medical Center, Boston, Mass. RESULTS: The ratio of peak fluorescence intensities of the neoplastic lesions to contralateral normal control mucosa were consistently different compared with these ratios in benign lesions or normal mucosa. These differences were seen in 2 distinct regions of the fluorescence spectrum with both of the excitation wavelengths, but were more obvious with the excitation wavelength of 410 nm. Using these differences, we were able to correctly diagnose 17 of the 19 lesions studied, with 2 false-positive results. CONCLUSIONS: Neoplastic oral mucosa shows consistent differences in autofluorescence spectral intensity and line shape when compared with the normal mucosa in the same individual. These early results show that fluorescence spectroscopy may represent a useful technique for noninvasive early diagnosis of cancer of the upper aerodigestive tract.

Feasibility of measuring blood glucose concentration by near-infrared Raman spectroscopy.

We report the determinations of glucose concentrations in human whole blood samples made using near-infrared Raman spectroscopy. Raman spectra of blood samples with above-physiological levels of glucose were acquired for 5 min through the wall of a cuvette via fiber optics. Partial least squares analysis was used to predict glucose concentrations in the samples. A root mean squared prediction error of 3.6 mM glucose was achieved with a correlation coefficient of 0.99 between reference and predicted values. This result is the first step in evaluating the potential of near-infrared Raman spectroscopy to perform blood glucose measurement with clinical accuracy. The technique is capable of measuring the concentration of other Raman-active blood constituents; as an example, bicarbonate was also measured. The method could eventually be useful for direct measurement of tissue analytes.

Absolute oil fluorescence conversion efficiency.

A conceptual method has been developed to deduce the characteristic infinite thickness absolute conversion efficiencies of oils from measured finite thickness film fluorescent returns. The sea water Raman signal is used as a reference in deducing the fluorescent efficiencies. The former can be obtained absolutely by measuring its temporal behavior, from which an absolute conversion efficiency of an oil can be deduced, without any a priori knowledge of the oil. The effects of signal-to-noise and background fluorescence on the accuracy of the fluorescent efficiency measurement are considered.

Infrared laser photography with silver-halide emulsion.

Infrared sensitization of photographic emulsion response to visible light is a useful technique for visualizing IR laser beams. In this work, it is demonstrated that this sensitization results from heating by the IR beam. Investigation of the dependence of sensitization by pulsed CO(2) laser (10.6-microm) preexposures on visible background density, IR fluence, visible wavelength, delay between exposures, and spatial frequency are described. Infrared fluences as low as 10 mJ/cm(2), a dynamic range greater than 100, and a resolution better than 50 lines/mm were observed. Suggestions for further improvements of the technique are made.

Unstable resonator mode control in a transverse flow dye laser.

Using suitable correcting optics, a 2x diffraction-limited beam has been obtained from a high power transverse flow dye laser. Beam divergence in the direction parallel to flow is controlled by a sound wave and may be compensated for externally.

Efficient cavity dumped dye laser.

A theoretical and experimental investigation of cavity dumped flashlamp pumped dye lasers is described. Theory predicts that pulse time compressions of the order of 10-20 can be obtained without loss in energy efficiency. Experimental confirmation was obtained with a coaxial flashlamp dye laser. Over 300 mJ of output was obtained in a 20-nsec pulse at 490 nm.

An enhanced algorithm for linear multivariate calibration.

We present a new method of linear multivariate calibration that can generate better prediction results than those obtained by partial least squares (PLS). This is accomplished by incorporating the spectrum of the desired species into the calibration procedure. The method combines the advantages of different standard methods and is therefore called hybrid linear analysis (HLA). In side-by-side tests using both simulated and experimental data, HLA produced lower prediction errors than PLS in all instances. We recommend HLA over PLS in situations where the spectrum of the desired species is available.

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.