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Background: The frequency of EMS runs, resuscitation attempts, & out of hospital cardiac arrest survival within a pandemic period has not been studied within the Detroit and metro Detroit region in Michigan. To analyze and study the consequences associated with the spread of COVID-19 within two Michigan counties, a retrospective review from two different medical control authorities (MCA) was completed. We hypothesized the number of out of hospital cardiac arrests and EMS runs to increase during the COVID-19 pandemic, while resuscitation attempts to decrease. Methods: This is a retrospective review study. Records from two MCAs, the Detroit East Medical Control Authority (DEMCA) and the Macomb County Medical Control Authority (MCMCA), were utilized and data was extracted and extrapolated. Each of these MCAs oversee Emergency Medical Services (EMS) within their respective counties. DEMCA provides coverage for the eastern region of Wayne County and MCMCA serves all of Macomb County. Runs, dead-on-scene numbers, & resuscitation attempts were each assessed from March through May of 2019 & 2020. Results: There were nearly double the number of EMS runs for out of hospital cardiac arrest during the 3 main months of the pandemic. In March through May 2020 there were 1756 runs with a mean age of 62.7 years vs. 988 runs during the same period in 2020 with a higher mean age of 65.1 years (p Conclusion: There was a 1.78 fold rise in out of hospital cardiac arrest EMS runs during the peak months of the pandemic in metro Detroit compared to the same period in 2019. Despite national concerns on the safety of resuscitation with Covid-19, a higher proportion of patients had resuscitation attempted during the pandemic.

Cancer of the pancreas (CaP) is a dismal, uncommon, systemic malignancy. This article updates an earlier experience of actual long-term survival of CaP in patients treated between 1991 to 2000, and reviews the literature. Survival is expressed as actual, not projected, survival.

Laser-induced fluorescence (LIF) and intrinsic fluorescence spectroscopy (IFS) have been used experimentally for diagnosing coronary atherosclerosis. In this study, we demonstrated the diagnostic superiority of IFS at 342-nm excitation (IFS342) versus LIF (LIF342) and described a protocol for head-to-head comparison of old (LIF) versus new (IFS) generations of similar diagnostic methods, labeled as “generational comparison model”. IFS342 and LIF342 were modeled with basis spectra of media, fibrous caps, and superficial foam cells and of their correspondent chemicals (elastin, collagen, and lipoproteins). The average accuracy and receiver operating characteristic area under the curve of IFS342 in single-, double-, and triple-parameter diagnostic algorithm iterations, geared toward identifying 84 atherosclerotic specimens from a group of 117 coronary segments, was 90% ± 1% and 0.87 ± 0.025, superior to LIF342 (84% ± 3% and 0.84 ± 0.016; P = 0.0002 and 0.02, respectively) in a generational comparison model.

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the non-enzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and non-glycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision - with a limit of detection that is nearly a factor of 15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multi-component mixtures and potentially in hemolysate and whole blood lysate samples.

The present invention relates to a full-field reflection phase microscope. In a preferred embodiment, the invention can combine low-coherence interferometry and off-axis digital holographic microscopy (DHM). The reflection-based DHM provides highly sensitive and a single-shot imaging of cellular dynamics while the use of low coherence source provides a depth-selective measurement. A preferred embodiment of the system uses a diffraction grating in the reference arm to generate an interference image of uniform contrast over the entire field-of-view albeit low-coherence light source. With improved path-length sensitivity, the present invention is suitable for full-field measurement of membrane dynamics in live cells with sub-nanometer-scale sensitivity.

Objectives: The protein components of low-density lipoprotein (LDL), oxidized LDL and proteoglycans such as versican contain tryptophan, an amino acid with characteristic fluorescence features at 308 nm excitation wavelength. We hypothesize that intrinsic fluorescence spectroscopy at 308 nm excitation wavelength (IFS308), a method suitable for clinical use, can identify coronary artery lesions with superficial foam cells (SFCs) and/or proteoglycans. Methods: We subjected 119 human coronary artery specimens to in vitro fluorescence and reflectance spectroscopy. We used 5 basis spectra to model IFS308, and extracted their contributions to each individual IFS308 spectrum. A diagnostic algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was built to identify specimens with SFCs and/or proteoglycans in their top 50m. Results: We detected SFCs and/or proteoglycans, such as versican or the glycosaminoglycan hyaluronan, in 24 fibrous cap atheromas or pathologic intimal thickening (PIT) lesions. An algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was able to identify these segments with 92% sensitivity and 80% specificity. Conclusion: We were able to establish a set of characteristic LDL, oxidized LDL, versican and hyaluronan fluorescence spectra, ready to be used for real-time diagnosis. The IFS308 technique detects SFCs and/or proteoglycans in fibrous cap atheromas and PIT lesions. SFCs and proteoglycans are histological markers of vulnerable plaques, and this study is a step further in developing an invasive clinical tool to detect the vulnerable atherosclerotic plaque. © 2010 Elsevier Ireland Ltd. All rights reserved.

Sample-to-sample variability has proven to be a major challenge in achieving calibration transfer in quantitative biological Raman spectroscopy. Multiple morphological and optical parameters, such as tissue absorption and scattering, physiological glucose dynamics and skin heterogeneity, vary significantly in a human population introducing non-analyte specific features into the calibration model. In this paper, we show that fluctuations of such parameters in human subjects introduce curved (non-linear) effects in the relationship between the concentrations of the analyte of interest and the mixture Raman spectra. To account for these curved effects, we propose the use of support vector machines (SVM) as a non-linear regression method over conventional linear regression techniques such as partial least squares (PLS). Using transcutaneous blood glucose detection as an example, we demonstrate that application of SVM enables a significant improvement (at least 30%) in cross-validation accuracy over PLS when measurements from multiple human volunteers are employed in the calibration set. Furthermore, using physical tissue models with randomized analyte concentrations and varying turbidities, we show that the fluctuations in turbidity alone causes curved effects which can only be adequately modeled using non-linear regression techniques. The enhanced levels of accuracy obtained with the SVM based calibration models opens up avenues for prospective prediction in humans and thus for clinical translation of the technology.

Objectives: We used reflectance and fluorescence spectroscopy to noninvasively and quantitatively distinguish benign from dysplastic/malignant oral lesions. We designed diagnostic algorithms to account for differences in the spectral properties among anatomic sites (gingiva, buccal mucosa, etc). Methods: In vivo reflectance and fluorescence spectra were collected from 71 patients with oral lesions. The tissue was then biopsied and the specimen evaluated by histopathology. Quantitative parameters related to tissue morphology and biochemistry were extracted from the spectra. Diagnostic algorithms specific for combinations of sites with similar spectral properties were developed. Results: Discrimination of benign from dysplastic/malignant lesions was most successful when algorithms were designed for individual sites (area under the receiver operator characteristic curve [ROC-AUC], 0.75 for the lateral surface of the tongue) and was least accurate when all sites were combined (ROC-AUC, 0.60). The combination of sites with similar spectral properties (floor of mouth and lateral surface of the tongue) yielded an ROC-AUC of 0.71. Conclusions: Accurate spectroscopic detection of oral disease must account for spectral variations among anatomic sites. Anatomy-based algorithms for single sites or combinations of sites demonstrated good diagnostic performance in distinguishing benign lesions from dysplastic/malignant lesions and consistently performed better than algorithms developed for all sites combined.

The physiological lag between blood and interstitial fluid (ISF) glucose is a major challenge for noninvasive glucose concentration measurements. This is a particular problem for spectroscopic techniques, which predominantly probe ISF glucose, creating inconsistencies in calibration, where blood glucose measurements are used as a reference. To overcome this problem, we present a dynamic concentration correction (DCC) scheme, based on the mass transfer of glucose between blood and ISF, to ensure consistency with the spectral measurements. The proposed formalism allows the transformation of glucose in the concentration domain, ensuring consistency with the acquired spectra in the calibration model. Taking Raman spectroscopy as a specific example, we demonstrate that the predicted glucose concentrations using the DCC-based calibration model closely match the measured glucose concentrations, while those generated with the conventional calibration methods show significantly larger deviations from the measured values. In addition, we provide an analytical formula for a previously unidentified source of limiting uncertainty arising in spectroscopic glucose monitoring from a lack of knowledge of glucose kinetics in prediction samples. A study with human volunteers undergoing glucose tolerance tests indicates that this lag uncertainty, which is comparable in magnitude to the uncertainty arising from noise and nonorthogonality in the spectral data set, can be reduced substantially by employing the DCC scheme in spectroscopic calibration.

The human red blood cell (RBC) membrane, a fluid lipid bilayer tethered to an elastic 2D spectrin network, provides the principal control of the cell’s morphology and mechanics. These properties, in turn, influence the ability of RBCs to transport oxygen in circulation. Current mechanical measurements of RBCs rely on external loads. Here we apply a noncontact optical interferometric technique to quantify the thermal fluctuations of RBC membranes with 3 nm accuracy over a broad range of spatial and temporal frequencies. Combining this technique with a new mathematical model describing RBC membrane undulations, we measure the mechanical changes of RBCs as they undergo a transition from the normal discoid shape to the abnormal echinocyte and spherical shapes. These measurements indicate that, coincident with this morphological transition, there is a significant increase in the membrane’s shear, area, and bending moduli. This mechanical transition can alter cell circulation and impede oxygen delivery.

OBJECTIVES: We used reflectance and fluorescence spectroscopy to noninvasively and quantitatively distinguish benign from dysplastic/malignant oral lesions. We designed diagnostic algorithms to account for differences in the spectral properties among anatomic sites (gingiva, buccal mucosa, etc). METHODS: In vivo reflectance and fluorescence spectra were collected from 71 patients with oral lesions. The tissue was then biopsied and the specimen evaluated by histopathology. Quantitative parameters related to tissue morphology and biochemistry were extracted from the spectra. Diagnostic algorithms specific for combinations of sites with similar spectral properties were developed. RESULTS: Discrimination of benign from dysplastic/malignant lesions was most successful when algorithms were designed for individual sites (area under the receiver operator characteristic curve [ROC-AUC],0.75 for the lateral surface of the tongue) and was least accurate when all sites were combined (ROC-AUC, 0.60). The combination of sites with similar spectral properties (floor of mouth and lateral surface of the tongue) yielded an ROC-AUC of 0.71. CONCLUSIONS: Accurate spectroscopic detection of oral disease must account for spectral variations among anatomic sites. Anatomy-based algorithms for single sites or combinations of sites demonstrated good diagnostic performance in distinguishing benign lesions from dysplastic/malignant lesions and consistently performed better than algorithms developed for all sites combined., National Institutes of Health (U.S) (R0I-CA097966), National Institutes of Health (U.S) (P4I-RR02594- 21)

Using quantitative phase microscopy, we have discovered a quadratic relationship between the radius R and the thickness t of helical ribbons that form spontaneously in multicomponent cholesterol–surfactant mixtures. These helical ribbons may serve as mesoscopic springs to measure or to exert forces on nanoscale biological objects. The spring constants of these helices depend on their submicroscopic thickness. The quadratic relationship ( R ∝ t 2 ) between radius and thickness is a consequence of the crystal structure of the ribbons and enables a determination of the spring constant of any of our helices solely in terms of its observable geometrical dimensions.

Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.

Cellular transformation is associated with a number of phenotypic, cell biological, biochemical and metabolic alterations. The detection and classification of morphological cellular abnormalities represents the foundation of classical histopathology and remains an important mainstay in the clinic. More recently, significant effort is being expended towards the development of noninvasive modalities for the detection of cancer at an early stage, when therapeutic interventions are highly successful. Methods that rely on the detection of optical signatures represent one class of such approaches that have yielded promising results. In our study, we have applied two spectroscopic imaging approaches to systematically identify in a quantitative manner the fluorescence and light scattering signatures of subcellular abnormalities that are associated with cellular transformation. Notably, we find that tryptophan images reveal not only intensity but also localization differences between normal and human papillomavirus immortalized cells, possibly originating from changes in the expression, 3D packing and organization of proteins and protein-rich subcellular organelles. Additionally, we detect alterations in cellular metabolism through quantitative evaluation of the NADH, FAD fluorescence and the corresponding redox ratio. Finally, we use light scattering spectroscopy to identify differences in nuclear morphology and subcellular organization that occur from the nanometer to the micrometer scale. Thus, these optical approaches provide complementary biomarkers based on endogenous fluorescence and scattering cellular changes that occur at the molecular, biochemical and morphological level. Since they obviate the need for staining and tissue removal and can be easily combined, they provide desirable options for further clinical development and assessment.

Objective— Foam cells perform critical functions in atherosclerosis. We hypothesize that coronary segments with superficial foam cells (SFCs) situated in a region of interest with a depth of 200 μm can be identified using intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS). This is a key step in our ongoing program to develop a spectroscopic technique for real-time in vivo diagnosis of vulnerable atherosclerotic plaque. Methods and Results— We subjected 132 human coronary segments to in vitro IFS and DRS. We detected SFCs in 13 thick fibrous cap atheromas and 8 pathologic intimal thickening (PIT) lesions. SFCs colocalized with accumulations of smooth muscle cells and proteoglycans, including hyaluronan ( P 40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively. Conclusion— Our combined IFS and DRS technique accurately detects SFCs in thick fibrous cap atheromas and PIT lesions. Because SFCs are associated with histological markers of plaque erosion, our spectroscopic technique could prove useful in identifying vulnerable plaques.

We present the first demonstration of in vivo collection of Raman spectra of breast tissue. Raman spectroscopy, which analyzes molecular vibrations, is a promising new technique for the diagnosis of breast cancer. We have collected 31 Raman spectra from nine patients undergoing partial mastectomy procedures to show the feasibility of in vivo Raman spectroscopy for intraoperative margin assessment. The data was fit with an established model, resulting in spectral-based tissue characterization in only 1 second. Application of our previously developed diagnostic algorithm resulted in perfect sensitivity and specificity for distinguishing cancerous from normal and benign tissues in our small data set. Significantly, we have detected a grossly invisible cancer that, upon pathologic review, required the patient to undergo a second surgical procedure. Had Raman spectroscopy been used in a real-time fashion to guide tissue excision during the procedure, the additional reexcision surgery might have been avoided. These preliminary findings suggest that Raman spectroscopy has the potential to lessen the need for reexcision surgeries resulting from positive margins and thereby reduce the recurrence rate of breast cancer following partial mastectomy surgeries. (Cancer Res 2006; 66(6): 3317-22)

Reflectance and fluorescence spectroscopies have shown great promise for early detection of epithelial dysplasia. We have developed a clinical reflectance spectrofluorimeter for multimodal spectroscopic diagnosis of epithelial dysplasia. This clinical instrument, the FastEEM, collects white light reflectance and fluorescence excitation-emission matrices (EEM's) within a fraction of a second. In this paper we describe the FastEEM instrumentation, designed for collection of multi-modal spectroscopic data. We illustrate its performance using tissue phantoms with well defined optical properties and biochemicals of known fluorescence properties. In addition, we discuss our plans to develop a system that combines a multi-spectral imaging device for wide area surveillance with this contact probe device.

Optical spectroscopic methods are playing an increasingly important role in medicine, and gastrointestinal endoscopy is likely to be one of the most important areas of impact of the new technology. This article will familiarize endoscopists with basic technologies of diagnostic spectroscopies, discuss the types of information they provide, and describe how they can be incorporated in endoscopic instruments. The discussion focuses on 4 fundamental light-tissue interactions: elastic scattering, absorption, fluorescence, and Raman scattering. The physical basis of each technique and its mode of implementation in endoscopic applications are presented. We discuss the limitations and advantages of each technique, as well as research that uses spectroscopic approaches for the diagnosis of gastrointestinal disease. Finally, we discuss future directions in spectral diagnostic endoscopy.

BACKGROUND Understanding the development and progression of head and neck squamous cell carcinoma is key in the quest for the early diagnosis and prevention of this type of malignancy. The current study correlated early biochemical and histologic changes in oral tissue with spectral features in fluorescence, reflectance, and light scattering spectra acquired in vivo to diagnose early stages of oral malignancies. METHODS A total of 91 tissue sites from 15 patients with varying degrees of malignancy (normal, dysplastic, and cancerous sites) and 8 healthy volunteers were analyzed with 3 spectroscopic techniques. Direct biochemical information regarding oral tissue native fluorophores was obtained with intrinsic fluorescence spectroscopy by fitting a linear combination of collagen and the reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence spectra to the intrinsic tissue fluorescence spectra excited with 337 nanometer (nm) and 358-nm laser light. Diffuse reflectance spectroscopy was used to provide information regarding tissue absorption and structure, such as hemoglobin concentration and stroma density, by measuring the wavelength-dependent absorption and scattering coefficients. By subtracting the diffusely reflected component from the measured reflectance, light scattering spectroscopy (LSS) information resulting from single backscattering from epithelial cell nuclei was obtained. LSS provides information concerning the size distribution of cell nuclei. RESULTS These optically extracted tissue parameters provide biochemical or structural information in vivo without the need for tissue excision, and can be used to diagnose tissue abnormalities. By combining the information provided by the three techniques, a method known as trimodal spectroscopy, a sensitivity and specificity of 96% and 96%, respectively, in distinguishing cancerous/dysplastic (mild, moderate, and severe) from normal tissue was achieved. In addition, the authors were able to distinguish dysplastic from cancerous tissue with a sensitivity of 64% and a specificity of 90%. CONCLUSIONS The results of the current study demonstrated that Trimodal spectroscopy is a highly sensitive and specific technique with which to diagnose tissue abnormalities. Cancer 2003;97:1681–92. © 2003 American Cancer Society. DOI 10.1002/cncr.11255

We have developed a portable multiexcitation dye laser system pumped by a 308 nm excimer laser, which can collect fluorescence spectra at 11 excitation wavelengths and a white light reflectance spectrum in less than 200 ms. The system is designed to be used in a hospital setting for in vivo tissue analysis based on fluorescence and reflectance spectroscopy. A specially designed optical fiber probe with SMA couplers facilitates easy alignment, important in a clinical setting. An intensified charge coupled device camera provides fast data acquisition with large signal-to-noise (S/N) ratio. The optical and electronic systems are controlled with LABVIEW, which is well suited for real time analysis, to extract diagnostic information. We present typical spectra that demonstrate the large excitation-emission wavelength range over which spectra can be acquired with very good S/N ratios.

Surface-enhanced Raman scattering (SERS) is a spectroscopic technique which combines modern laser spectroscopy with the exciting optical properties of metallic nanostructures, resulting in strongly increased Raman signals when molecules are attached to nanometre-sized gold and silver structures. The effect provides the structural information content of Raman spectroscopy together with ultrasensitive detection limits, allowing Raman spectroscopy of single molecules. Since SERS takes place in the local fields of metallic nanostructures, the lateral resolution of the technique is determined by the confinement of the local fields, which can be two orders of magnitude better than the diffraction limit. Moreover, SERS is an analytical technique, which can give information on surface and interface processes. SERS opens up exciting opportunities in the field of biophysical and biomedical spectroscopy, where it provides ultrasensitive detection and characterization of biophysically/biomedically relevant molecules and processes as well as a vibrational spectroscopy with extremely high spatial resolution. The article briefly introduces the SERS effect and reviews contemporary SERS studies in biophysics/biochemistry and in life sciences. Potential and limitations of the technique are briefly discussed.

OBJECTIVE: The objective of this study was to assess the potential of 3 spectroscopic techniques (intrinsic fluorescence, diffuse reflectance, and light scattering) individually and in combination (trimodal spectroscopy) for the detection of cervical squamous intraepithelial lesions. STUDY DESIGN: The study was conducted with 44 patients who underwent colposcopy for the evaluation of an abnormal Papanicolaou smear. Fluorescence and reflectance spectra were collected from colposcopically normal and abnormal sites and analyzed to extract quantitative information about tissue biochemistry and morphologic condition. This information was compared with histopathologic classification, and diagnostic algorithms were developed and validated with the use of logistic regression and cross-validation. RESULTS: Diagnostically significant differences exist in the composition of fluorescing biochemicals, the scattering properties, and the epithelial cell nuclear morphology of cervical squamous intraepithelial lesions and non–squamous intraepithelial lesions. Trimodal spectroscopy is a superior tool for the detection of cervical squamous intraepithelial lesions than any 1 of the techniques alone. CONCLUSION: Trimodal spectroscopy has the potential to improve the in vivo detection of precancerous cervical changes. (Am J Obstet Gynecol 2002;186:374-82.)

Ultrasensitive Raman measurements in single living cells are possible through exploiting the effect of surface-enhanced Raman scattering (SERS). Colloidal gold particles (60 nm in size) that are deposited inside cells as “SERS-active nanostructures” result in strongly enhanced Raman signals of the native chemical constituents of the cells. Particularly strong field enhancement can be observed when gold colloidal particles form colloidal clusters. The strongly enhanced Raman signals allow Raman measurements of a single cell in the 400–1800 cm−1 range with 1-μm lateral resolution in relatively short collection times (1 second for one mapping point) using 3–5 mW near-infrared excitation. SERS mapping over a cell monolayer with 1-μm lateral resolution shows different Raman spectra at almost all places, reflecting the very inhomogeneous chemical constitution of the cells. Colloidal gold supported Raman spectroscopy in living cells provides a tool for sensitive and structurally selective detection of native chemicals inside a cell, such as DNA and phenylalanine, and for monitoring their intracellular distributions. This might open up exciting opportunities for cell biology and biomedical studies.

Pancreatic cancer is a systemic disease for most patients. Operations with the intent to cure may be done safely (mortality, < 3%) with shorter hospital stays. Surgery has been minimally effective as a long-term cure. Endoscopic palliation of jaundice is becoming standard practice. Common sense dictates a defined and clear role for surgery (i.e., strict patient selection criteria). Surgery is of great value for small, localized lesions; of clear value as palliative therapy when nonoperative measures fail; and perhaps best applied after neoadjuvant chemoradiotherapy. Surgery is just part of the armamentarium available to treat pancreatic cancer. Novel systemic therapies, including chemotherapy, immunotherapy, and so-called targeted therapies, are becoming increasingly valuable in the management of this systemic disease and are discussed in detail in other articles in this issue.

Raman spectroscopy has the potential to provide real-time, in situ diagnosis of breast cancer during needle biopsy or surgery via an optical fiber probe. Understanding the chemical/morphological basis of the Raman spectrum of breast tissue is a necessary step in developing Raman spectroscopy as a tool for in situ breast cancer diagnosis. To understand the relationship between the Raman spectrum of a sample of breast tissue and its disease state, near-infrared Raman spectroscopic images of human breast tissue were acquired using a confocal microscope. These images were then compared with phase contrast and hematoxylin- and eosin-stained images to develop a chemical/morphological model of breast tissue Raman spectra. This model fits macroscopic tissue spectra with a linear combination of basis spectra derived from spectra of the cell cytoplasm, cell nucleus, fat, β-carotene, collagen, calcium hydroxyapatite, calcium oxalate dihydrate, cholesterol-like lipid deposits and water. Each basis spectrum represents data acquired from multiple patients and, when appropriate, from a variety of normal and diseased states. The model explains the spectral features of a range of normal and diseased breast tissue samples, including breast cancer. It can be used to relate the Raman spectrum of a breast tissue sample to diagnostic parameters used by pathologists. Copyright © 2002 John Wiley & Sons, Ltd.

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.

Once a surgeon has determined that a patient with malignant pancreatic or periampullary disease is a candidate for operative intervention, cardiac assessment is an important next step before surgery. Often, these patients are elderly, malnourished, or have significant comorbid medical problems, including chronic obstructive pulmonary disease and diabetes mellitus, which increase the risk for noncardiac surgery. Preoperative preparation can maximize these organ systems, but the fear of a major perioperative cardiac event exists for every surgeon. That 40% of patients undergoing noncardiac surgery in the United States each year have, or are at risk for, coronary artery disease supports this concern. 8 Cardiac complications are a leading cause of death in the intraoperative and postanesthesia period. Cardiology consultation should be obtained to help identify patients at high risk for perioperative morbidity and mortality, especially among candidates for extensive surgical resection. Congestive heart failure, hypertension, and atrial fibrillation can increase perioperative risk. Clinical detection and adequate management of these problems can decrease risks in the postoperative period. Identification of asymptomatic left ventricular dysfunction by preoperative echocardiography can alert the physician to potential problems of fluid management to avoid postoperative pulmonary edema. Symptomatic critical aortic stenosis should be corrected before any elective surgery; however, in most patients, evaluation is performed primarily to predict the probability of a perioperative myocardial ischemic event. Although an electrocardiogram is a routine first screen, determining which patients need further noninvasive testing is less clear. Certainly, it is not cost-effective to subject every patient to a battery of tests. Risk classification based on clinical assessment has been developed and validated to help answer this question. Two major indices are available. The first classification, the Eagle Index, 2 gives 1 point to each of the following categories: 1A history of myocardial infarction or angina 2A Q wave on preoperative ECG 3Diabetes mellitus requiring drug therapy 4Age of more than 70 years 5A history of ventricular arrhythmia requiring therapy The second index, the Revised Cardiac Risk Index, 6 as described by Lee and associates, assigns 1 point to each of the following variables: 1High-risk surgery 2A history of ischemic heart disease as manifested by a history of myocardial infarction, a positive exercise test, current complaints of ischemic chest pain, the use of nitrate therapy, or an ECG with Q waves 3A history of congestive heart failure 4A history of cerebral vascular disease 5Insulin therapy for diabetes mellitus 6Preoperative creatinine level of more than 2.0 mg/dL If no Eagle Index points are present, the patient is considered to be at low risk; 1 or 2 points classifies the patient as an intermediate risk; more than 2 points, high risk. In the Revised Cardiac Risk Index, high risks include class III and class IV patients having two risk factors or more than two risk factors, respectively. Both classifications advocate the use of noninvasive testing in the intermediate-risk group. Lower-risk patients had a 3.1% risk for a perioperative myocardial event; the intermediate group, a 15% risk; and the high-risk group, a 50% risk. Intermediate-risk patients who were negative for ischemia on their workup had a risk factor of just 3.2%, similar to that of the low-risk group. Those who were positive had a risk of 29.6% of having a perioperative myocardial event. L'Italien et al 7 validated these results in their studies. Persantine-thallium screening provided no stratification for low and high-risk patients based on their clinical model. For patients in the intermediate risk category, 80% were classified as low risk after the study, and only 20%, as high risk. Basic noninvasive testing for risk stratification includes the ECG stress test, which is conducted in patients who can exercise and have normal ST segments at rest. 10 Poor exercise capacity and the development of ECG changes consistent with myocardial ischemia, particularly if it occurs at a rate less than 75% of predicted maximal heart rate is associated with a high risk for perioperative ischemic events. Patients who achieve greater than 75% of their predicted maximal heart rate without ST segment changes are at low risk. Those who achieve a greater than 75% maximal heart rate and show ischemic changes are at intermediate risk. Many patients are unable to exercise or have an abnormal ECG at rest, rendering an ECG response to exercise nondiagnostic. In these patients, pharmacologic nuclear perfusion imaging using thallium or dobutamine echocardiography can be used to detect myocardial ischemia. 3,4,11,13 Today, practice guidelines are available that support the concept of risk stratification based on a clinical risk index. 1,5 Patients at low risk need no additional testing. Those at high risk and intermediate risk with large zones of myocardial ischemia by noninvasive testing should undergo angiography and revascularization as indicated by angiographic findings before undergoing major intra-abdominal surgery. Findings requiring revascularization include left main trunk disease or triple vessel disease with left ventricular dysfunction. Coronary intervention in high-risk patients identified by myocardial perfusion scintigraphy may improve outcomes in major, noncardiovascular surgery. 14 Although revascularization in high-risk coronary patients seems to improve the long-term outcome of these patients, no prospective, randomized trials show that coronary artery surgical intervention in these patients reduces short-term perioperative ischemic complications. This has led to the use of β-blockade in patients undergoing major noncardiac surgery. 12 In one randomized trial, high-risk patients, defined as having an abnormal dobutamine stress echocardiogram, were randomized to receive β-blockade versus placebo. The group on β-blockade had a 3.4% prevalence of a perioperative myocardial ischemic event compared with a 33.9% prevalence in the placebo group. This remarkable reduction in risk was noted and the trial was halted early by an independent safety committee. Today, β-blockade has been used not only with increasing frequency in high-risk surgical patients but also in conjunction with coronary stenting when used as a method for coronary revascularization. To diminish the risk for stent thrombosis, intensive antiplatelet therapy is needed for at least 4 weeks. If possible, elective surgery should be delayed during this period. Based on the available literature and the authors' personal experience, the following recommendations are suggested for the evaluation and care of potential cardiac patients facing major pancreatic surgery. 1Patients with an unstable coronary syndrome should undergo angiography and appropriate revascularization, as indicated, before any elective surgery. 2Patients with clinical indications for revascularization (independent of their noncardiac condition) should undergo revascularization before noncardiac surgery. 3Patients with strongly positive noninvasive tests, such as dobutamine stress echocardiography, stress ECG, or nuclear perfusion imaging, should undergo angiography and revascularization as appropriate. 4Patients with symptomatic critical aortic stenosis should undergo aortic valve replacement before elective surgery. 5Noninvasive testing should be limited to intermediate-risk patients stratified by clinical criteria. High-risk patients should undergo angiography and appropriate revascularization before elective surgery, although the current literature offers no prospective study validating that such an intervention would reduce perioperative events. Low-risk patients require no further testing. 6Patients who have had revascularization in the previous 5 years, and those who have had noninvasive testing within the past 2 years without evidence of clinical ischemia, require no further preoperative testing. 7All clinically determined intermediate- and high-risk patients should receive β-blockade perioperatively unless contraindicated, such as in the setting of asthma, sinus bradycardia, and second- and third-degree arteriovenous blockade. 9 Following these guidelines, a concise recommendation can be given to the surgeon concerning the risks of surgical intervention for individual patients. These evaluations are vital to clinicians when deciding whether to perform surgery on a high-risk patient. The risks of operative intervention must be balanced with the outlook that each patient has in terms of his or her existing pancreatic disease.

Background: We have previously shown that Raman spectroscopy can be used for chemical analysis of intact human coronary artery atherosclerotic lesions ex vivo without tissue homogenization or extraction. Here, we report the chemical analysis of individual cellular and extracellular components of atherosclerotic lesions in different stages of disease progression in situ using Raman microspectroscopy. Methods: Thirty-five coronary artery samples were taken from 16 explanted transplant recipient hearts, and thin sections were prepared. Using a high-resolution confocal Raman microspectrometer system with an 830-nm laser light, high signal-to-noise Raman spectra were obtained from the following morphologic structures: internal and external elastic lamina, collagen fibers, fat, foam cells, smooth muscle cells, necrotic core, β-carotene, cholesterol crystals, and calcium mineralizations. Their Raman spectra were modeled by using a linear combination of basis Raman spectra from the major biochemicals present in arterial tissue, including collagen, elastin, actin, myosin, tropomyosin, cholesterol monohydrate, cholesterol linoleate, phosphatidyl choline, triolein, calcium hydroxyapatite, calcium carbonate, and β-carotene. Results: The results show that the various morphologic structures have characteristic Raman spectra, which vary little from structure to structure and from artery to artery. The biochemical model described the spectrum of each morphologic structure quite well, indicating that the most essential biochemical components were included in the model. Furthermore, the biochemical composition of each structure, indicated by the fit contributions of the biochemical basis spectra of the morphologic structure spectrum, was very consistent. Conclusion: The Raman spectra of various morphologic structures in normal and atherosclerotic coronary artery may be used as basis spectra in a linear combination model to analyze the morphologic composition of atherosclerotic coronary artery lesions.

Background: Recent studies have shown that chemical composition and morphology, rather than anatomy (degree of stenosis), determine atherosclerotic plaque instability and predict disease progression. Current clinical diagnostic techniques provide accurate assessment of plaque anatomy, but have limited capability to assess plaque morphology in vivo. Here we describe a technique for a morphology-based diagnosis of atherosclerosis in the coronary arteries using Raman spectroscopy that can potentially be performed in vivo using optical fiber technology. Methods: Raman tissue spectra were collected from normal and atherosclerotic coronary artery samples in different stages of disease progression (n=165) from explanted transplant recipient hearts (n=16). Raman spectra from the elastic laminae (EL), collagen fibers (CF), smooth muscle cells (SMC), adventitial adipocytes (AA) or fat cells, foam cells (FC), necrotic core (NC), cholesterol crystals (CC), β-carotene containing crystals (β-C), and calcium mineralizations (CM) were used as basis spectra in a linear least squares-minimization (LSM) model to calculate the contribution of these morphologic structures to the coronary artery tissue spectra. Results: We developed a diagnostic algorithm that used the fit-contributions of the various morphologic structures to classify 97 coronary artery samples in an initial calibration data set as either nonatherosclerotic, calcified plaque, or noncalcified atheromatous plaque. The algorithm was subsequently tested prospectively in a second validation data set, and correctly classified 64 (94%) of 68 coronary artery samples. Conclusions: Raman spectroscopy provides information about the morphologic composition of intact human coronary artery without the need for excision and microscopic examination. In the future, it may be possible to use this technique to analyze the morphologic composition of atherosclerotic coronary artery lesions and assess plaque instability and disease progression in vivo.

We present a novel instrument for imaging the angular distributions of light backscattered by biological cells and tissues. The intensities in different regions of the image are due to scatterers of different sizes. We exploit this to study scattering from particles smaller than the wavelength of light used, even when they are mixed with larger particles. We show that the scattering from subcellular structure in both normal and cancerous human cells is best fitted to inverse power-law distributions for the sizes of the scattering objects, and propose that the distribution of scattering objects may be different in normal versus cancerous cells.

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.

A sensitive ultraviolet resonance Raman (UVRR) system, which integrates bulk and microscopic capabilities, is described for use with human tissue. The system incorporates a quasi-continuous tunable laser excitation source in the range 200± 300 nm, a liquid nitrogencooled charge-coupled device (CCD), a single grating spectrometer, dielectric longpass laser line rejection ® lters, and cooled specimen holders to minimize bio- and photo-degradation. The system was used on normal human colon tissue, and was compared to our previously used Nd:YAG-based system. Spectra with higher signal-tonoise ratio (S/N) than has previously been achieved, and with less than 5% photobleaching, are presented. The present system outperforms our previous system by improving the S/N in bulk normal colon spectra by a factor of greater than 10. We show for the ® rst time UVRR spectra collected from the various microscopic regions of human colon in a thin section preparation, and the line shapes from the different microscopic morphological components are related to the bulk colon spectra. At 251 nm the epithelial cells and the lamina propria are the major contributors to the UVRR spectrum of bulk colon tissue. Our data suggest that combined bulk and microscopic UVRR spectroscopy of human tissue could provide a valuable tool for bioassay and histochemistry. Index Headings: Ultraviolet resonance Raman spectroscopy; Human colon; Quantitative histology.

In the Raman effect, incident light is inelastically scattered from a sample and shifted in frequency by the energy of its characteristic molecular vibrations. Since its discovery in 1927, the effect has attracted attention from a basic research point of view as well as a powerful spectroscopic technique with many practical applications. The advent of laser light sources with monochromatic photons at high flux densities was a milestone in the history of Raman spectroscopy and resulted in dramatically improved scattering signals (for a general overview of modern Raman spectroscopy, see refs 1-5). In addition to this so-called spontaneous or incoherent Raman scattering, the development of lasers also opened the field of stimulated or coherent Raman spectroscopies, in which molecular vibrations are coherently excited. Whereas the intensity of spontaneous Raman scattering depends linearly on the number of probed molecules, the coherent Raman signal is proportional to the square of this number (for an overview, see refs 6 and 7). Coherent Raman techniques can provide interesting new opportunities such as vibrational imaging of biological samples,8 but they have not yet advanced the field of ultrasensitive trace detection. Therefore, in the following article, we shall focus on the spontaneous Raman effect, in the following simply called Raman scattering. Today, laser photons over a wide range of frequencies from the near-ultraviolet to the near-infrared region are used in Raman scattering studies, allowing selection of optimum excitation conditions for each sample. By choosing wavelengths which excite appropriate electronic transitions, resonance Raman studies of selected components of a sample or parts of a molecule can be performed.9 In the past few years, the range of excitation wavelengths has been extended to the near-infrared (NIR) region, in which background fluorescence is reduced and photoinduced degradation from the sample is diminished. High-intensity NIR diode lasers are easily available, making this region attractive for compact, low cost Raman instrumentation. Further, the development of low noise, high quantum efficiency multichannel detectors (chargecoupled device (CCD) arrays), combined with highthroughput single-stage spectrographs used in combination with holographic laser rejection filters, has led to high-sensitivity Raman spectrometers (for an overview on state-of-the-art NIR Raman systems, see ref 10). As we shall show in section 2, the nearinfrared region also has special importance for ultrasensitive Raman spectroscopy at the singlemolecule level. As with optical spectroscopy, the Raman effect can be applied noninvasively under ambient conditions in almost every environment. Measuring a Raman spectrum does not require special sample preparation techniques, in contrast with infrared absorption spectroscopy. Optical fiber probes for bringing excitation laser light to the sample and transporting scattered light to the spectrograph enable remote detection of Raman signals. Furthermore, the spatial and temporal resolution of Raman scattering are determined by the spot size and pulse length, respectively, of the excitation laser. By using a confocal microscope, Raman signals from femtoliter volumes (∼1 μm3) can by observed, enabling spatially resolved measurements in chromosomes and cells.11 Techniques such as multichannel Hadamard transform Raman microscopy12,13 or confocal scanning Fourier transform Raman microscopy14 allow generation of high-resolution Raman images of a sample. Recently, Raman spectroscopy was performed using near-field optical microscopy.15-17 Such techniques overcome the diffraction limit and allow volumes significantly smaller than the cube of the wavelength to be investigated. In the time domain, Raman spectra can be measured on the picosecond time scale, providing information on short-lived species such as excited 2957 Chem. Rev. 1999, 99, 2957−2975

Surface-enhanced hyper-Raman scattering and surface-enhanced anti-Stokes Raman scattering were studied as potential tools for non-linear single-molecule Raman spectroscopy. Experiments were performed using near-infrared excitation on crystal violet adsorbed on colloidal silver or gold clusters. Strong enhancement factors on the order of 1020 were inferred from hyper-Raman scattering experiments on colloidal silver. Such extremely high enhancement factors overcome the inherently weak nature of the effect, and surface-enhanced hyper-Raman scattering appears on comparable intensity levels as surface-enhanced Raman scattering. Surface-enhanced anti-Stokes Raman scattering starts from vibrational levels, that are populated by the very strong surface-enhanced Raman process. Thus, the anti-Stokes Raman scattering signal depends quadratically on the excitation laser intensity. For the first time, surface-enhanced anti-Stokes and Stokes Raman scattering was detected from single molecules on colloidal gold clusters.

We present a nonparametric method of analysis of Raman spectra of coronary artery tissue to classify atherosclerotic lesions. The method correlates the principal component scores of the Raman spectra with the tissue pathology. A data set composed of 97 samples of human coronary artery was used to develop the diagnostic algorithm, and a second data set composed of 68 samples was then used to test this algorithm prospectively. The results show that the algorithm can accurately classify coronary artery tissue into three classes: nonatherosclerotic, noncalcified plaque, and calcified plaque. The accuracy of this classification scheme is comparable to that previously achieved by means of a biochemical analysis of the Raman spectra using the same data.

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.

Background: Previous in vitro studies showed that autofluorescence images of colonic mucosa collected endoscopically can be used to detect dysplasia with high sensitivity. This method is extended to the collection of fluorescence images of adenomatous polyps in vivo. Methods: Fluorescence images were collected during colonoscopy in 30 patients. A total of 12 adenomatous and 6 hyperplastic polyps were identified. A fiberoptic excitation probe, located in the instrument channel of the colonoscope, delivered 300 mW of near-ultraviolet light at λex = 351 and 364 nm. Mucosal fluorescence in the spectral bandwidth between 400 and 700 nm was imaged, processed, and displayed with various likelihoods of associated dysplasia. Results: Adenomatous polyps exhibited decreased fluorescence intensity compared with adjacent mucosa with normal appearance. With the fluorescence threshold set to 80% of the average intensity of normal mucosa, a sensitivity of 83% for dysplasia identification was achieved. All hyperplastic polyps were correctly identified as being non-dysplastic. Optimal identification of dysplastic regions was obtained with the colonoscope oriented at a near-normal angle of incidence to the polyps. At higher angles of incidence, artifacts caused by illumination shadows were introduced. Conclusions: The dysplasia associated with adenomatous polyps can be identified in vivo by fluorescence imaging with high sensitivity, thus demonstrating the potential to guide endoscopic procurement of biopsy specimens. (Gastrointest Endosc 1999;49:447-55.)

We report an in situ method of probing the structure of living epithelial cells, based on light scattering spectroscopy with polarized light. The method makes it possible to distinguish between single backscattering from uppermost epithelial cells and multiply scattered light. The spectrum of the single backscattering component can be further analyzed to provide histological information about the epithelial cells such as the size distribution of the cell nuclei and their refractive index. These are valuable quantities' to detect and diagnose precancerous changes in human tissues.

In visualizing transparent biological cells and tissues, the phase contrast microscope and its related techniques have been a cornerstone of nearly every cell biology laboratory. However, phase contrast methods are inherently qualitative and lack in 3-D imaging capability. We introduce a novel tomographic microscopy for quantitative three-dimensional mapping of refractive index in live cells and tissues using a phase-shifting laser interferometric microscope with variable illumination angle.

In agreement with previous results reported for colloidal silver clusters, effective surface-enhanced Raman cross sections of about 10-16 cm2 per molecule, corresponding to enhancement factors on the order of 10 14, have also been obtained for molecules attached to colloidal gold clusters. Spatially isolated nearly spherical colloidal gold particles of about 60 nm size show maximum enhancement factors on the order of 103 at 514 nm excitation, close to the single plasmon resonance. The enhancement factor increases by eleven orders of magnitude when colloidal gold clusters are formed by aggregation of the gold colloids and when near-infrared excitation is applied. The large effective surface-enhanced Raman cross section has been estimated by a straightforward method based on steady-state population redistribution due to the pumping of molecules to the first excited vibrational state via the strongly enhanced Raman process. Our experimental finding confirms the important role of colloidal clusters for extremely large surface-enhanced Raman scattering (SERS) enhancement factors. Simultaneously, it suggests colloidal gold clusters as a substrate for high-sensitivity surface-enhanced Raman scattering, which can provide an enhancement level sufficient for Raman single molecule detection. Due to its chemical inactivity, gold might have some advantages compared to silver, particularly in biomedical spectroscopy.

The present invention relates to systems and methods for quantitative three-dimensional mapping of refractive index in living or non-living cells, tissues, or organisms using a phase-shifting laser interferometric microscope with variable illumination angle. A preferred embodiment provides tomographic imaging of cells and multicellular organisms, and time-dependent changes in cell structure and the quantitative characterization of specimen-induced aberrations in high-resolution microscopy with multiple applications in tissue light scattering.

Surface-enhanced Raman scattering (SERS) at an extremely high enhancement level opens up interesting and new spectroscopic possibilities. The effect combines the sensitivity of fluorescence spectroscopy with the high structural information content of Raman spectroscopy, and can be used for single molecule detection and identification. This paper reports single molecule detection and identification of ‘non-absorbing’ molecules in colloidal silver solutions using near-infrared excited surface-enhanced Stokes and anti-Stokes Raman scattering. SERS enhancement factors of the order of 1014–1015 or, in other words, effective Raman cross-sections between 10-16 and 10-15 cm2/molecule result in a significant transfer of ground state population to the first excited vibrational state due to the strong Raman process. This allows the observation of v=1 to v=2 (‘hot’) vibrational transitions in SERS additionally to v=0 to v=1 transitions ‘normally’ probed in a Raman experiment. © 1998 John Wiley & Sons, Ltd.

This report describes surface-enhanced Stokes and anti-Stokes Raman scattering of molecules in aqueous colloidal silver solution using non-resonant near-infrared excitation. We demonstrate that extremely large surface-enhanced Raman cross sections of the order of 10−16 cm2 per molecule can be combined with favorable conditions for excitation and collection of Raman scattered light provided by a Raman microscope to achieve single molecule sensitivity. Surface-enhanced Raman spectroscopy will be compared with fluorescence spectroscopy as a tool for single molecule detection.

Nonresonant Raman cross sections of $\ensuremath{\sim}{10}^{\ensuremath{-}16} {\mathrm{cm}}^{2}$ per molecule are shown to be related to surface-enhanced Raman scattering (SERS) on colloidal silver clusters at near-infrared (NIR) excitation. The enhancement is found to be independent of cluster sizes between 100 nm and 20 \ensuremath{\mu}m. These experimental findings demonstrate that NIR SERS on colloidal silver clusters is an excellent technique for single molecule detection that is applicable for a broad range of molecules including ``colorless'' biomolecules, for example nucleotides in DNA sequencing. As an example, we present the detection of a single adenine molecule without any labeling based on its intrinsic surface-enhanced Raman scattering.

Purpose : Near-infrared Raman spectroscopy provides an important new means of analyzing the chemical composition of the arterial wall. The objective of this study was to show that Raman spectroscopy can be used to evaluate the lipid and calcium salt contents of human peripheral arteries. The results extend a recently developed Raman-based method for analyzing the chemical composition of coronary arteries. Methods and Results : We studied 167 segments of carotid and femoral artery wall in various pathologic states. The Raman spectra from these samples was accurately modeled. The resulting chemical concentrations were compared with the amounts of cholesterol and calcium mineral determined at histologic evaluation by an experienced cardiovascular pathologist. Strong correlations between spectroscopic measurements and morphologic findings were demonstrated and validated the applicability of the method to peripheral arteries. Conclusions : Raman spectroscopy can provide reliable histochemical information about peripheral and coronary arteries. Such information may help identify rupture-prone plaques before the onset of symptoms and allow aggressive and directed intervention. Accurate knowledge of the chemical composition of a lesion may be useful in selecting the most appropriate treatment. (J Vasc Surg 1998;27:710-9.)

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.

Single-molecule Raman spectroscopy of a cyanine dye in aqueous silver colloidal solution with the use of surface-enhanced Raman scattering at near-infrared excitation (NIR-SERS) is reported. A characteristic Poisson distribution of SERS signals due to the Brownian motion of single dye molecule-loaded silver particles reflects the probability of finding 0, 1, or 2 1,1'-diethyl-2,2'cyanine (PIC) molecules in the probed volume during an actual measurement and is evidence that single-molecule detection by SERS has been achieved. Spectra measured in 1 s collection time with 100 mW nonresonant 830 nm excitation provide a clear “fingerprint” of a single PIC molecule by showing its typical Raman lines between 700 and 1700 cm−1. Single-molecule Raman signals are also detected for the first time at the anti-Stokes side of the excitation laser. Effective Raman cross sections for PIC of ∼10−16 cm2 per molecule can be inferred from the ratio between “pumped” anti-Stokes and Stokes signals.