The Stokes-shifted emission spectra were measured for various photoactive biomolecules such as tryptophan, collagen, NADH and flavin in aqueous solution and tissue. Information is obtained on the molecular activity in the tissue. This new approach allows for the extraction of information not obtained from excitation and/or fluorescence spectroscopy for a single spectral scan.

To estimate the intrinsic fluorescence intensity decay of a compound, the excitation light pulse must be deconvolved from the measured fluorescence pulse trace. The most commonly used deconvolution method is the multiexponential least-square iterative reconvolution (LSIR) technique. A variant of LSIR in which the intrinsic fluorescence intensity decay is expressed as an expansion on the discrete time Laguerre basis, was recently introduced. In this study, the performance of the Laguerre deconvolution technique was successfully tested with simulated and fluorescence standard data. It was also demonstrated that the Laguerre deconvolution presents a number of advantages over the classical multiexponential LSIR, including less expensive computational resolution, and the property to generate a unique set of expansion coefficients highly correlated with the intrinsic lifetimes. A novel method for concentration estimation based on the analysis of the Laguerre expansion coefficients was also proposed and successfully applied to different fluorescence standard mixtures, performing even better (error<2%) than more traditional methods of spectral analysis, such as PCR (error<7%) and PLS (error<10%). These findings suggest that the use of Laguerre expansion coefficients represents an alternative nonparametric approach to characterize and discriminate biological systems, in terms of their spectral and lifetime characteristics.

Development of AD is associated with cerebrovascular deposition of amyloid beta (Aβ) as well as a progressive increase in vasular collagen content. Both AΒ and collagen are naturally fluorescent compounds when exposed to UV light. We analyzed autofluorescence emitted from brain tissue samples and isolated brain resistance vessels harvested postmortem from patients with Alzheimer's disease (AD) and age-matched controls. Fluorescence emission, excited at 355 nm with an Nd:YAG laser, was measured using a fiber-optic based fluorescence spectroscopic system for tissue analysis. Significantly higher values of fluorescence emission intensity (P<0.001) in the spectral region from 465 to 490 nm were detected in brain resistance vessel samples from AD patients compared to the normal individuals. Results from western blot analysis showed elevated levels of type I and type III collagen, and reduced levels of type IV collagen in resistance vessels from AD patients, compared to control samples. In addition, using direct scanning of the cortical suface for fluoresxcence emission by the laser-induced fluorescence spectroscopy system we detected a significantly (P<0.05) higher level of apoptosis in AD brain tissue compared to age-matched controls. Fluorescence emission analysis (FEA) appears to be a sensitive technique for detecting structural changes in AD brain tissue.

Noninvasive evaluation of tissue viability of donor kidneys used for transplantation is an issue that current technology is not able to address. In this work, we explore optical spectroscopy for its potential to assess the degree of ischemic damage in kidney tissue. We hypothesized that ischemic damage to kidney tissue will give rise to changes in its optical properties which in turn may be used to asses the degree of tissue injury. The experimental results demonstrate that the autofluorescence intensity of the injured kidney is decreasing as a function of time exposed to ischemic injury. Changes were also observed in the NIR light scattering intensities most probably arising from changes due to injury and death of the tissue.

The carotenoids lycopene and beta-carotene are powerful antioxidants in skin and are thought to act as scavengers for free radicals and singlet oxygen. The role of carotenoid species in skin health is of strong current interest. We demonstrate the possibility to use Resonance Raman spectroscopy for fast, non-invasive, highly specific, and quantitative detection of beta-carotene and lycopene in human skin. Analyzing Raman signals originating from the carbon-carbon double bond stretch vibrations of the carotenoid molecules under blue and green laser excitation, we were able to characterize quantitatively the relative concentrations of each carotenoid species in-vivo. In the selective detection, we take advantage of different Raman cross-section spectral profiles for beta-carotene and lycopene molecules, and obtain a quantitative assessment of individual long-chain carotenoid species in the skin rather than their cumulative levels. Preliminary dual-wavelength Raman measurements reveal significant differences in the carotenoid composition of different subjects. The technique holds promise for rapid screening of carotenoid compositions in human skin in large populations and may be suitable in clinical studies for assessing the risk for cutaneous diseases.

The low-frequency torsional modes of tryptophan were measured by terahertz time-domain spectroscopy (THz-TDS). It was found that there are two dominated torsional vibrational modes at around 1.435 and 1.842 THz. The origin of the observed torsional vibrations are assigned to the chain and ring of the tryptophan molecule.

Here, we propose a new method to enhance the sensitivity of the reflectance spectrum to the scattering feature of the superficial tissue layer. This method is based on multiple discriminant analysis (MDA) in the eigen subspace of the spectrum. Considering the application of scattering imaging, we evaluated this method by performing multispectral imaging of two-layered tissue phantoms. A color map converted from the spectral reflectance well corresponds to variations in the size of the scatter in the first layer. In order to confirm our proposed method works well under more realistic conditions, we performed the computational simulations of the light propagation in the tissue. We used the simulation model combined with the Monte Carlo and the Mie scattering. Its conditions like the slab geometry and the particle distribution of the cell nucleus were estimated by the image measuring of pathological slices. Results on simulations show the possibility of enhancing the sensitivity of the reflectance spectrum to the scattering feature of the superficial tissue layer.

The scattering centers in cells are not spheres, however, in most modeling of light transport, the scattering centers are assumed to be spherical. For example, in Monte Carlo simulations a Mie or Henyey-Greenstein phase function is often used. It is known that an elliptical particle will have a different phase function than a spherical particle. In particular there are differences in the phase functions for scattering polarized light. To examine how these changes in phase function affect light transport in tissue, we have developed a Monte Carlo code for light transport that uses elliptical scatterers. The phase functions are calculated using a T-matrix code and the propagation of polarized photons is performed in a manner analagous to that used by Bartel and Hielscher. Our initial results indicate that for narrow particle distributions the difference in shape can cause large differences in the intensity and polarization properties of the diffusely reflected light. For a mixture of particle sizes, however, there is a much smaller difference in the properties of the diffusely scattered light. Results are presented for both narrow and broad distributions of scatter sizes relevant to tissue.

More than two million cases of nonmelanoma skin cancers are diagnosed every year. Therefore, there is a strong need for practical, reliable, rapid, and precise methods for tumor delineation, to guide surgery and other treatments of skin cancer. Once developed, such methods may be useful for squamous cell carcinomas of other organs. Non-invasive optical imaging techniques including polarization sensitive reflectance and fluorescence imaging were evaluated for the demarcation of nonmelanoma skin tumors. Thick freshly excised tumor specimens obtained from Mohs surgery were used for the experiments. Imaging was performed using linearly polarized incident light in the visible and near infrared spectral range from 577 nm to 750 nm. Non-toxic absorbing and fluorescent dyes (Toluidine Blue O, Methylene Blue) were employed to enhance tumor contrast in the images. The images were acquired using the remitted light polarized in the directions parallel and perpendicular to the polarization of incident light. Reflectance and fluorescence polarization images were evaluated. The data were processed and analyzed for dependence of the remitted light polarization on the tissue type (cancerous/normal). The data obtained so far from fresh tumor specimens in vitro using dye-enhanced polarized light reflectance, and exogenous fluorescence polarization imaging suggest that optical mapping can become a valuable guidance tool in nonmelanoma cancer surgery.

Coherent backscattering (CBS) is a photon weak-localization phenomenon that gives rise to an enhanced backscattering of light by random media. Although this effect has been previously studied in nonbiological media, there have been only few attempts to use CBS for diagnosis and characterization in living tissue. Here we report spectroscopic CBS measurements (low-coherence CBS spectroscopy) by combining broadband illumination and low-coherence detection. We demonstrate that low-coherence CBS spectroscopy substantially simplifies CBS measurements in biological tissue and enables depth-resolved spectroscopic analysis of CBS. Low-coherence CBS spectroscopy may find important applications in probing biological tissue where depth-selective measurements are crucial. As an example of the potential of CBS for tissue diagnosis, we show that low-coherence CBS spectroscopy can be used to detect the earliest, previously undetectable, preadenomatous stages of colorectal carcinogenesis.

Optical molecular imaging of living tissues could potentially allow tissue characterization and disease diagnosis with high resolution, non-invasively and at a low cost [1]. The capability to image multiple molecular targets simultaneously is particularly important. However, currently this task cannot be achieved, due to the broad spectral responses (~80-200 nm) of current optical contrast agents, which limit the number of markers that can be used simultaneously to, typically, not more than three [1-6]. Therefore, ideal nanoparticle-labels for multi-color multi-marker detection should exhibit sharp resonances, whose spectral position can be controlled. We hereby describe a new concept that utilizes resonant light-scattering spectroscopy of multi-layered metallic nanospheres to achieve the labeling of multiple targets simultaneously. Our model of light-scattering predicts that multilayered metallic-dielectric nanoshells exhibit tunable ultra-sharp resonance peaks with widths as narrow as 10 nm. By varying the materials and size ratios between different layers, the position of the resonance peaks can be easily controlled, which may enable detection of more than 10 different labels simultaneously by recording the spectra of resonant light scattering in the visible range. Conjugated with molecular probes, such as antibodies, these novel structured nanospheres may enable previously unattainable multi-label optical molecular imaging.

An instrument for imaging skin lesions in four different polarization states and at four wavelengths is described. The instrument is based upon a polarization subtraction method that can be used to extract weakly scattered light and remove surface reflections, without the need to resort to matching fluid and glass plates. Monte Carlo simulations of a layered medium are used to demonstrate the sensitivity of the measurements to the underlying medium. Ideally one would like to extract the absorption, scattering and layer thickness of the medium. However, the problem is ill-conditioned and some prior knowledge of the lesion properties will be necessary for successful implementation.

The NASA Smart Probe employs multiple microsensors, together with fuzzy logic and neural networks, to provide a unique tissue "signature" in real time. We here review the Smart Probe concept and summarize early data from small animal studies on tissue identification. Recent clinical information gathered from women undergoing biopsy for suspected breast cancer by the NASA licensee, BioLuminate Inc., is also presented. The sensors employed in the Smart Probe for breast cancer include electrical impedance and optical spectroscopy (both broadband or white light, and laser light (infrared and blue/fluorescence)). Data are acquired 100 times per second; a typical breast "biopsy" typically generates 500 MB of data. The multiparameter breast cancer probe-one millimeter in diameter-can clearly differentiate normal breast, benign lesions, and breast carcinoma.

The significance of normal mitochondrial function in cellular energy homeostasis as well as its involvement in acute and chronic neurodegenerative disease was reviewed recently (Nicholls & Budd. Physiol Rev. 80: 315-360, 2000). Nevertheless, monitoring of mitochondrial function in vivo and real time mode was not used by many investigators and is very rare in clinical practice. The main principle tool available for the evaluation of mitochondrial function is the monitoring of NADH fluorescence. In order to interpret correctly the changes in NADH redox state in vivo, it is necessary to correlate this signal to other parameters, reflecting O₂ supply to the brain. Therefore, we have developed and applied a multiparametric optical monitoring system, by which microcirculatory blood flow and hemoglobin oxygenation is measured, together with mitochondrial NADH fluorescence. Since the calibration of these signals is not in absolute units, the simultaneous monitoring provide a practical tool for the interpretation of brain functional state under various pathophysiological conditions. The monitoring system combines a time-sharing fluorometer-reflectometer for the measurement of NADH fluorescence and hemoglobin oxygenation as well as a laser Doppler flowmeter for the recording of microcirculatory blood flow. A combined fiber optic probe was located on the surface of the brain using a skull cemented cannula. Rats and gerbils were exposed to anoxia, ischemia and spreading depression and the functional state of the brain was evaluated. The results showed a clear correlation between O₂ supply/demand as well as, energy balance under the various pathophysiological conditions. This monitoring approach could be adapted to clinical monitoring of tissue vitality.

The development of a technique that gives early diagnosis and is non-invasive is of crucial importance for public health. Raman spectroscopy is a technique that can full fill these requirements. The main goal of this work was to use the FT-Raman spectroscopy to differentiate between normal skin and the squamous cell carcinoma (SCC) tissues in vitro. The samples used in this study were collected by traditional human biopsy of skin tissue. The samples removed from the patients were washed in physiologic serum and frozen in liquid nitrogen. The FT-Raman device used was the RFS 100- Bruker, with a 1064nm from the Nd:YAG as an excitation source. After the Raman measurement the samples were submitted for histopathological study for comparation. The Raman spectra in the normal tissue showed the presence of vibrational bands in 860 cm^-1 and 939 cm^-1 with higher intensity than in the carcinoma spectra. These modes were assigned to the vibration of proline and hydroxiproline. The shift region of 1555 to 1560 cm^-1 showed a difference of intensity to the samples of squamous cell carcinoma, which were attributed to the nucleic acid.

Skin injury caused by chemicals substances as the carrageenan produces a local inflammatory reaction involving the liberation of mediators that leads to an increase in vascular permeability and, consequently, edema formation. The vascular permeability can be evaluated by measuring the amount of some extravasating specific dyes. The Evans blue dye is recommended due to its systemic effect and non-toxicity to the organism. That dye binds to the plasma albumin and emits radiation when excited, allowing for spectroscopic monitoring of the edema. In this study, the amount of extravasating plasma albumin in the site of the carrageenan-induced edema in Wistar rats is evaluated by fluorescence spectroscopy. The intensity of the Evans blue dye fluorescence signal for different edema evolution times is compared to the 125I labeled albumin data obtained with a g-counter. A dye laser (458 nm) was used as the fluorescence excitation source. The fluorescence intensity was taken at the 680 nm peak of the dye spectral emission. The spectroscopic data shows the dye emission intensity growing with the settling up of the edema and decreasing as the tissue recovers from the inflammatory stimulus. A good correlation between the spectroscopic and the g-counter data was obtained, which suggests that the Evans blue dye fluorescence is a promising technique for the qualitative and quantitative analysis of edema dynamics.

The aim of this study is to explore the possibility of correlating hemodynamic changes and neural activities in the brain by using an integrated system combining Near Infrared Spectroscopy (NIRS) and electroencephalographic activity (EEG). We present brain hemodynamic changes and EEG recordings obtained from four volunteers during the performance of two different sequential thumb-finger opposition tasks, with and without a related mental activity. The optical and electrical signals were recorded simultaneously on the subject forehead. The coupling of the two systems could be useful to demonstrate correlation between cognitive paradigms and hemodynamic signals.