Intrinsic Tissue Fluorescence Research Articles

Contribution of autofluorescence from intracellular proteins in multiphoton fluorescence lifetime imaging

Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex. Particularly when utilizing two-photon excitation, as conventionally employed in MPM. This increases the possibility for spectral crosstalk and incorrect assignment of the origin of the FLIM signal. Here we demonstrate that in keratinocytes, proteins such as keratin may interfere with the signal usually assigned to NADH in MPM-FLIM by contributing to the lifetime component at 1.5 ns. This is supported by a change in fluorescence lifetime distribution in KRT5- and KRT14-silenced cells. Altogether, our results suggest that the MPM-FLIM data originating from cellular autofluorescence is far more complex than previously suggested and that the contribution from other tissue constituents should not be neglected—changing the paradigm for data interpretation in this context.

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions.

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Capabilities of fluorescent spectroscopy in diagnosis of tumors of the ent-organs

Introduction. Diagnosis verification in patients with malignant tumors developed in the context of chronic inflammation is technically complex with a high percentage of false negative results of histological examination. None of the methods provides sufficiently accurate results necessary for ruling out or confirming a diagnosis.The study objective is to test a new patented method of pre- and intraoperative differential diagnosis of oncological pathology of the ent-organs using the "InSpektr-M" hardware and software system.Materials and methods. The study included 30 patients with suspected malignant tumors of the larynx. The study excluded patients with previously verified diagnosis of malignant tumor of the larynx. A diagnostic probe emitting laser light with wavelength 350 nm and radiation power density 25 mW/cm2 was introduced through an additional channel of a fiberscope for a period of time sufficient for excitation of intrinsic tissue fluorescence (from 10-6 to 10-9 ms). Fluorescence intensity in the range of wavelengths between 600 and 650 nm was measured. This intensity was conditionally labelled "epithelial proliferative activity index". Spectrometric analysis of the intact points allowed to evaluate normal values for an individual patient; in the areas with the maximal increase in the index, targeted biopsy was performed.Results. Values obtained during examination of various types of tumors (epithelial and non-epithelial) are different while tumor location has a negligible effect. In patients with malignant tumors containing non-squamous epithelium, increased protoporphyrin IX and decreased FAD fluorescence were observed in the direction from intact tissues to the center of the suspected tumor; in patients with malignant tumors containing squamous epithelium: increased protoporphyrin IX fluorescence with maximum at 635 nm and dynamic increase in FAD fluorescence in the direction from intact tissues to the tumor center.Conclusion. Fluorescent spectroscopy can be used for pre- and intraoperative differential diagnosis of squamous and non-squamous cancers of the ENT-organs. Further studies for establishment of reference values of fluorescence intensity in intact and affected tissues are necessary.

Wide-field optical spectroscopy system integrating reflectance and spatial frequency domain imaging to measure attenuation-corrected intrinsic tissue fluorescence in radical prostatectomy specimens.

The development of a multimodal optical imaging system is presented that integrates endogenous fluorescence and diffuse reflectance spectroscopy with single-wavelength spatial frequency domain imaging (SFDI) and surface profilometry. The system images specimens at visible wavelengths with a spatial resolution of 70 µm, a field of view of 25 cm2 and a depth of field of ∼1.5 cm. The results of phantom experiments are presented demonstrating the system retrieves absorption and reduced scattering coefficient maps using SFDI with <6% reconstruction errors. A phase-shifting profilometry technique is implemented and the resulting 3-D surface used to compute a geometric correction ensuring optical properties reconstruction errors are maintained to <6% in curved media with height variations <20 mm. Combining SFDI-computed optical properties with data from diffuse reflectance spectra is shown to correct fluorescence using a model based on light transport in tissue theory. The system is used to image a human prostate, demonstrating its ability to distinguish prostatic tissue (anterior stroma, hyperplasia, peripheral zone) from extra-prostatic tissue (urethra, ejaculatory ducts, peri-prostatic tissue). These techniques could be integrated in robotic-assisted surgical systems to enhance information provided to surgeons and improve procedural accuracy by minimizing the risk of damage to extra-prostatic tissue during radical prostatectomy procedures and eventually detect residual cancer.

Tissue Intrinsic Fluorescence Spectra-Based Digital Pathology of Liver Fibrosis by Marker-Controlled Segmentation.

Tissue intrinsic emission fluorescence provides useful diagnostic information for various diseases. Because of its unique feature of spectral profiles depending on tissue types, spectroscopic imaging is a promising tool for accurate evaluation of endogenous fluorophores. However, due to difficulties in discriminating those sources, quantitative analysis remains challenging. In this study, we quantitatively investigated spectral-spatial features of multi-photon excitation fluorescence in normal and diseased livers. For morphometrics of multi-photon excitation spectra, we examined a marker-controlled segmentation approach and its application to liver fibrosis assessment by employing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. We formulated a procedure of internal marker selection where markers were chosen to reflect typical biochemical species in the liver, followed by image segmentation and local morphological feature extraction. Image segmentation enabled us to apply mathematical morphology analysis, and the local feature was applied to the automated classification test based on a machine learning framework, both demonstrating highly accurate classifications. Through the analyses, we showed that spectral imaging of native fluorescence from liver tissues have the capability of differentiating not only between normal and diseased, but also between progressive disease states. The proposed approach provides the basics of spectroscopy-based digital histopathology of chronic liver diseases, and can be applied to a range of diseases associated with autofluorescence alterations.

Tissue intrinsic fluorescence recovering by an empirical approach based on the PSO algorithm and its application in type 2 diabetes screening.

In order to reduce the influence of scattering and absorption on tissue fluorescence spectra, after tissue fluorescence and diffuse reflectance in different tissue optical properties were simulated by the Monte Carlo method, a tissue intrinsic fluorescence recovering algorithm making use of diffuse reflectance spectrum was developed. The empirical parameters in the tissue intrinsic fluorescence recovering algorithm were coded as a particle in the solution domain, the classification performance was defined as the fitness, and then a particle swarm optimization (PSO) algorithm was established for empirical parameters optimization. The skin autofluorescence and diffuse reflectance spectra of 327 subjects were collected in Anhui Provincial Hospital. The skin intrinsic autofluorescence spectra were recovered by using the empirical approach and the integration area of the spectra were calculated as fluorescence intensity. Receiver operating characteristic (ROC) analysis for fluorescence intensity was applied to evaluate the classification performance in type 2 diabetes screening. In addition, a support vector machine (SVM) method was implemented to improve the performance of the classification. The results showed that the sensitivity and specificity were 32% and 76% respectively, and the area under the curve was 0.54 before recovering, while the sensitivity and specificity were 72% and 86% respectively, and the area under the curve was 0.86 after recovering. Furthermore, the sensitivity and specificity increased to 83% and 86% respectively when using linear SVM while 84% and 88%, respectively, when using nonlinear SVM. The results indicate that using the tissue fluorescence spectrum recovery algorithm based on PSO can improve the application of tissue fluorescence spectroscopy effectively.

组织固有荧光光谱复原算法及其在糖尿病筛查中的应用研究

组织固有荧光光谱复原算法及其在糖尿病筛查中的应用研究

基于粒子群优化算法的生物组织固有荧光光谱复原方法

基于粒子群优化算法的生物组织固有荧光光谱复原方法

Con A conjugated to Europium(III) cryptate as a new histological tool for prostate cancer investigation using confocal microscopy

Lectins are carbohydrate recognition proteins that can be used as probes to reveal the glycosylation state of cells. They frequently have been used for diagnostic and prognostic cancer studies. For fluorescence based analysis, lectins commonly are conjugated to fluorescein isothiocyanate (Con A-FITC); however, this molecule loses its fluorescence quickly. We conjugated Europium cryptate to Con A (Con A-cryp-Eu) for use as a histochemical luminescent probe to recognize glucose/mannose residues in benign prostatic hyperplasia and prostatic carcinoma tissues, and used confocal microscopy instead of commercial Con A-FITC. Tissues were treated with Evans blue to suppress intrinsic tissue fluorescence before incubation with Con A-cryp-Eu or Con A-FITC. Con A-cryp-Eu exhibited hemagglutinating activity. Con A-cryp-Eu showed the same binding pattern as Con A-FITC in prostate stroma and gland cells. Staining was strong in benign prostate hyperplasia and prostate carcinoma tissues. Con A-cryp-Eu probe stained glucose/mannose residues in prostatic carcinoma more intensely than Con A-FITC. Furthermore, staining with Con A-cryp-Eu showed greater fluorescence intensity than Con A-FITC and the emission of Con A-cryp-Eu was more stable than the Con A-FITC for seven days under the same storage conditions. Maintenance of the luminescent properties and the binding pattern of Con A-cryp-Eu favor its use as an auxiliary histochemistry probe for prostatic tissue studies.

Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs

Multiphoton microscopy of cleared tissue has previously been demonstrated to generate large three-dimensional (3D) volumetric image data on entire intact mouse organs using intrinsic tissue fluorescence. This technique holds great promise for performing 3D virtual biopsies, providing unique information on tissue morphology, and guidance for subsequent traditional slicing and staining. Here, we demonstrate the use of fluorescence lifetime imaging in cleared organs for achieving molecular contrast that can reveal morphologically distinct structures, even in the absence of knowledge of the underlying molecular source. In addition, we demonstrate the power of multimodal imaging, combining multiphoton fluorescence, second harmonic generation, and lifetime imaging to reveal exceptional morphological detail in an optically cleared mouse knee.

Multiphoton Microscopy of Entire Intact Mouse Organs

Three-dimensional datasets from tissue biopsies may provide critical morphological information that is not readily obtained from traditional approaches to histology using thin physical sections of tissue. Multiphoton microscopy (MPM) provides optical sectioning with penetration into highly scattering materials, ready excitation of intrinsic tissue fluorescence, and access to nonlinear signals such as second harmonic generation (SHG). However, the penetration depth of MPM is typical limited to ∼200 microns in many tissues. We present MPM of entire intact, fixed and optically cleared mouse organs. Clearing of tissue is typically incomplete for large tissue samples, however, MPM has sufficient tolerance to scattering to image entire mouse organs. Using macro lenses with 5x magnification and 0.5 numerical aperture enables the generation of high resolution, large field-of-view datasets with imaging depths of several millimeters, sufficient to generate 3D image sets of mouse intestine, heart, lung, brain and other organs.

Quantification of Mlck Activation in Arteries of Living Mice That Express a Genetically Encoded Fret-Based Biosensor

FRET (Forster resonance energy transfer)-based sensors are powerful tools for understanding physiological mechanisms at the molecular level. Here, we used transgenic mice that express, specifically in smooth muscle, a FRET-based, exogenous myosin light chain kinase biosensor (‘exMLCK’) to observe for the first time changes in [Ca2+/Calmodulin] and MLCK activation within arteries in vivo (‘intra-vital FRET imaging’). exMLCK biosensor mice were anesthetized (1.5% isoflurane) and placed on the stage of a ‘macro’ epi-fluorescence microscope (Olympus MVX 10) equipped with an image splitter and a digital CCD camera. Arterial blood pressure (AP) was recorded. Femoral or mesenteric arteries were exposed through cutaneous incisions. Quantification of exMLCK FRET in vivorequires precise assessment and removal of tissue intrinsic fluorescence and extraneous sources of light. Intrinisic fluorescence (of isolated arteries) was negligible in comparison to exMLCK fluorescence. In vivo, background fluorescence was minimized by dissection of surrounding tissues and the use of an appropriate field diaphragm. Artery diameter was determined using edge detection and spatially averaged exMLCK FRET ratio (CFP/YFP) was determined from regions of interest. In the basal state (mean AP of ∼90 mm Hg), average exMLCK FRET ratio in femoral artery walls was 1.8 to 1.9, similar to that obtained by us in isolated arteries. Application of the α1-adrenoceptor agonist, phenylephrine (PE) directly to an artery increased exMLCK FRET ratio transiently to a peak of about 2.2 and caused a local vasoconstriction of ∼33%, without changing AP. Intra-venous application of PE elevated AP and caused smaller changes in exMLCK FRET ratio and artery diameter (than direct application). In conclusion, quantitative intra-vital FRET imaging in arteries of transgenic animals is feasible and will permit observation of specific molecular events in tissues of living animals. (AHA, NIH-HL078870).

Components of intrinsic fluorescence revealed by Metabolic Modulation Matrix in isolated rat cardiac myocytes

Aim: Recent developments in multi-wavelength fluorescence lifetime spectroscopy brought simultaneous measurement of fluorescence spectra and lifetimes in complex samples, namely cell and tissue intrinsic fluorescence. However, emission spectra of endogenous fluorophores are often superimposed in broad bands over the 400-600 nm wavelength region, making their separation particularly difficult. We therefore present a newly developed method of separation. Based on Metabolic Modulation Matrix approach, this method is built on evaluation of spectral modulation of endogenous fluorescence following changes in the cell metabolic state. Methods: Spectral fingerprints of time-resolved fluorescence are determined in isolated cardiomyocytes after excitation by 375-nm pulsed picosecond laser diode using SPC-830 TCSPC measurement system (Becker-Hickl on Zeiss Axiovert 200). Metabolic modulation was induced by respiratory chain and/or oxidative stress regulators. The number and profiles of the most significant spectral components were identified by time-resolved area-normalized emission spectroscopy and principal component analysis. Results: The Metabolic Modulation Matrix approach applied to time-resolved spectroscopy data in living cardiac cells demonstrated the presence of at least 3 significant spectrally-distinct components of NAD(P)H fluorescence corresponding to: i) NAD(P)H in water-like environment, ii) NAD(P)H in restricted-motion environment and iii) a flavin-type component. Lifetimes, revealed by fluorescence decay analysis showed values of τ1∼0.4 ± 0.1 ns and τ 2∼1.0 ± 0.2 ns for component i), τ ∼3.2 ± 0.8 ns, for component ii), and τ∼2.5-5 ns for component iii). Conclusions: Presented Metabolic Modulation Matrix concept, in conjunction with spectrally-resolved fluorescence lifetime detection of the cell intrinsic fluorescence, is a promising, highly versatile tool for quantitative assessment of oxidative metabolism in living cells.

Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals

A wide variety of pathological pathways may result in age-related macular degeneration. Because of its complexity, there is no comprehensive model of the disease yet. One key feature is the accumulation of the autofluorescent pigment lipofuscin in the retinal pigment epithelium (RPE). Thus, we developed an organotypic perfusion culture model of the porcine ocular fundus, generating lipofuscin under exposure to blue light and hydrogen peroxide. Porcine fundi (choroid, Bruch's membrane, RPE, and retina) were explanted in toto, transferred into a perfusion culture chamber, perfused with cell culture medium and kept at 37 degrees C. Free radical stress was induced by supplementation of H(2)O(2), and/or the specimens were exposed to blue light, or kept untreated as controls. After a culture period of 7 days, the specimens were subject to microscopic inspection, histology, fluorescence microscopy, and measurement of fluorescence spectra as well as fluorescence decay times. Histology showed atrophic ganglion cells and rod outer segments. All other tissue structures were morphologically intact. Compared to the controls, RPE and retina exposed to light showed increased fluorescence, which was shifted towards shorter wavelengths. The fluorescence spectra and decays resembled that of lipofuscin granules isolated from human donor eyes. HPLC analysis revealed the abundance of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2E), its precursor products, as well as two new, green-emitting fluorophores. Porcine ocular fundi were successfully preserved in an organotypic perfusion culture for 7 days, and exhibited remarkable autofluorescence after light and free radical exposure, making the model suitable for investigations of lipofuscinogenesis.

Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells

Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.

Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy*♦

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism.

Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen® and Hoechst 33258

Objective To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreen®, for determining DNA content in human cartilage.Methods Human articular and nasal septal cartilage explants were digested using proteinase K. Portions of sample digest were analysed for intrinsic and dye-enhanced fluorescence with either Hoechst 33258 or PicoGreen®.Results Intrinsic tissue fluorescence in both articular and septal cartilage increased with age and was prominent at wavelengths used for Hoechst 33258 but relatively low at wavelengths used for PicoGreen®. The relative contribution of intrinsic fluorescence to total dye-enhanced fluorescence of human cartilage was markedly greater for Hoechst 33258 (19–57%) than for PicoGreen® (2–7%). Thus, in many situations, DNA in human cartilage can be assayed using PicoGreen® without the need to correct for intrinsic cartilage fluorescence. The enhancement of fluorescence by each dye was found to be specific for DNA, as shown by fluorescence spectra, >90% sensitivity to DNase, and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in the absence or presence of proteinase K digested human cartilage, once intrinsic cartilage fluorescence was subtracted. PicoGreen® was more sensitive for assaying DNA (0.9ng DNA/ml) than Hoechst 33258 (6ng DNA/ml) and can also be used in a microplate reader.Conclusion PicoGreen® can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage. Copyright 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption

The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.

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

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). 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. 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. 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.

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.