Resonant Raman detection of macular pigment levels in the living human retina.

We have used resonant Raman scattering as a novel, noninvasive in vivo optical technique to measure the concentration of macular carotenoid pigments in the living human retina. Using a backscattering geometry and resonant molecular excitation in the visible, we measure the Raman peaks that originate from the single- and double-bond stretch vibrations of the p -conjugated molecule's carbon backbone. The Raman signals scale linearly with carotenoid content, whereas the required laser excitation is well under safety limits for macular exposure. The Raman technique is objective and quantitative and may lead to a new method for rapid screening of carotenoid pigment levels in large human populations that are at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the United States.

Resonance Raman detection of carotenoid antioxidants in living human tissues.

We have used resonance Raman scattering as a novel noninvasive optical technology to measure carotenoid antioxidants in living human tissues of healthy volunteers. By use of blue-green laser excitation, clearly distinguishable carotenoid Raman spectra superimposed on a fluorescence background are obtained. The Raman spectra are obtained within less than a minute, and the required laser light exposure levels are well within safety standards. Our technique can be used for rapid screening of carotenoid levels in large populations and may have applications for assessing antioxidant status and the risk for diseases related to oxidative stress.

Non-invasive raman spectroscopic detection of carotenoids in human skin.

Carotenoids are thought to play a significant part in the skin's anti-oxidant defense system, and may help prevent malignancy. Inability to measure skin carotenoid content readily has, however, made it difficult to establish the relationship between carotenoid concentration and the occurrence of cutaneous malignancy. We have measured in vivo carotenoid concentration using a noninvasive optical method, Raman spectroscopy. To validate our instrumentation, abdominoplasty skin was evaluated by both Raman spectroscopy and high-performance liquid chromatography determination for carotenoid content. Evaluation of the Raman signal in specific carotenoid solutions was also performed. Precision of Raman measurements within skin sites, within subjects, and between subjects was measured. Sensitivity of the method was evaluated as a function of anatomical region and the distribution of carotenoids within the stratum corneum. Lastly, we evaluated the Raman signal in actinic keratosis and basal cell carcinoma lesions and perilesional skin and compared this with region-matched sites in healthy subjects. Our results indicate that the Raman scattering method reflects the presence of carotenoids in human skin and is highly reproducible. Evaluation of five anatomical regions demonstrated significant differences in carotenoid concentration by body region with the highest carotenoid concentration noted in the palm. Comparison of carotenoid concentrations in basal cell carcinomas, actinic keratosis, and their perilesional skin demonstrate a significantly lower carotenoid concentration than in region-matched skin of healthy subjects. These results represent the first evidence that carotenoid concentration in the skin correlate with the presence or absence of skin cancer and precancerous lesions.

Raman detection of macular carotenoid pigments in intact human retina.

PURPOSE: To develop and test a novel noninvasive optical technique suitable for the objective measurement of macular carotenoid levels in human retina. METHODS: A resonance Raman scattering apparatus was constructed to measure carotenoid levels in flat-mounted human retinas and eyecups and in experimental animal eyes. Light from an argon laser was used to resonantly excite the electronic absorption of the carotenoid pigments, and scattered light was collected and analyzed by a Raman spectrometer. After carotenoid Raman measurements were completed on the retinal samples, macular carotenoid levels were determined by high-performance liquid chromatography (HPLC). RESULTS: Carotenoid resonance Raman scattering proved to be a highly sensitive and specific method for the noninvasive measurement of macular pigments in the human retina. Signal strength scaled linearly with actual macular carotenoid content as measured by HPLC. Our apparatus was also used to record resonance Raman signals from xanthophyll carotenoids stored in the retinal pigment epithelium of intact frog eyes. CONCLUSIONS: This new noninvasive optical method will facilitate studies of ocular carotenoid distributions and their role in degenerative diseases of the eye and may allow for the rapid screening of carotenoid levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness in the elderly in the United States. A prototype clinical instrument is under development.

Application of charge-coupled device technology for measurement of laser light and fluorescence distribution in tumors for photodynamic therapy.

Laser and fluorescence light distributions with applications for photodynamic therapy were measured in mouse tumors using a non-invasive electronic optical imaging system. The system consists of a liquid-nitrogen-cooled, charge-coupled-device (CCD) array camera under computer control with 576 x 384 detection elements having dimensions of 23 microns x 23 microns. The available dynamic range of the array is approx. 10(3), and the effective wavelength range is 400-1000 nm. An interstitially placed cylindrical diffusing optical fiber was used to provide tumor illumination. The light distribution pattern from the fiber was determined by immersing the cylindrical diffusing tip in a fluorescing solution and recording the emission image. Fluorescence imaging facilitates an accurate measurement of light intensity distribution while avoiding problems associated with the directional nature of other detection methods used with diffusing fibers. Radiation-induced fibrosarcoma tumors on C3H mice were grown to about 1 cm diameter for in vivo recording of light distribution from the tumor volume and for determination of effective light penetration distance at 18 wavelengths in the range 458-995 nm. Endogenous tumor fluorescence and Photofrin II fluorescence intensity were measured over the wavelength range 585-725 nm to investigate the possible application of CCD imaging technology for drug distribution measurements. Model experiments were begun to evaluate the relative importance of potential distortions of light distribution measurements using this approach.