Biochemical fingerprints of human papillomavirus infection and cervical dysplasia using cervical fluids: Spectral pattern investigation

The Pap smear is the primary screening tool for invasive cervical cancer resulting from a persistent infection with oncogenic human papillomavirus (HPV); however, there are the problems such as the inability to distinguish between HPV infection and cervical dysplasia and a low sensitivity remain. We present preliminary findings of a label‐free method to detect and classify HPV infection and cervical dysplasia using human cervical fluids. Three experimental groups, defined as normal, HPV‐positive, and cervical dysplasia, were evaluated through their Raman spectral patterns for noise‐independence, high reproducibility, and uniformity. Clinical diagnosis was performed through liquid‐based cervical cytology, HPV test, and cervical histologic examination. Healthy cervical fluids showed a strong Raman intensity at 877 cm^?1 (symmetric C–C stretching), and at 963 cm^?1 (phosphate), compared to a reference Raman peak at 1003 cm^?1 (phenylalanine symmetric ring breath). The HPV‐positive cervical fluids showed a strong intensity of a Raman peak at 1448 cm^?1 corresponding to C–H deformation vibration mode and the highest similarity between the central and ring zones among the three groups. The cervical dysplasia fluids showed the presence of strong peaks compared to the control and HPV‐positive groups. In addition, different Raman spectra were acquired according to HPV type. Therefore, all ranges of cervical fluid‐induced Raman spectra could be used to detect the presence of cervical pre‐cancer. Raman peak‐gated assessment provides a label‐free and nondestructive tool for the clinical diagnosis of HPV infection and cervical precancerous changes.     

Label‐free optical detection of age‐related and diabetic oxidative damage in human aqueous humors

In this study, we investigate the biochemical characteristics of oxidative stress in age‐related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm^?1. We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C–O stretching or C–O–H bending modes), AG (adenine and guanine), PRO‐AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α‐helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C–O stretching, C–O–H bending modes in carbohydrates (1043 cm^?1), and the nucleic acids adenine and guanine (1352 cm^?1), respectively. DR‐related oxidative damage was identified by PRO‐AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm^?1) and amide I α‐helix protein structure (1656 cm^?1), respectively. AMD‐related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress‐induced AMD and DR diseases. Therefore, Raman‐based label‐free optical detection is effective for detecting the presence of age‐related or diabetic oxidative damage in aqueous humor.     

Characterization of an ultraviolet irradiation chamber to monitor molecular photodegradation by Raman spectroscopy

A homemade ultraviolet chamber is reported to induce photochemical changes with characterization by Raman spectroscopy. The equipment has compartments for ultraviolet-A (UV-A) (8 lamps of 8 W) and for UV-A + ultraviolet-B (UV-B) irradiation (4 lamps of 26 W and 1 lamp of 15 W). The irradiance was measured 3, 5, and 10 cm from the light sources. The maximum irradiance was obtained at 3 cm (UV-A: 2.66 mW/cm² and UV-A + UV-B: 4.30 mW/cm²). The chamber internal temperature was stabilized at 30°C after 1 hr of operation with an internal relative humidity of approximately 45%. 10% Collagen was irradiated with UV-A at 2.0 mW/cm² for 3 hr with changes in Raman peaks at 1253, 1271, 1453, and 1660 cm^?1 indicating changes in conformation. 5% Atenolol was irradiated with UV-A + UV-B at 4.30 mW/cm² for 8 hours with changes to Raman peaks at 822, 1186, 1206, 1248, and 1618 cm^?1. A commercial insect repellent was irradiated with UV-A + UV-B at 4.30 mW/cm² for 8 hr and decreases in Raman intensity were observed at 526, 690, 1003, and 1606 cm^?1 due to degradation of N, N-diethyl-m-toluamide. The results demonstrate proper operation of the irradiation chamber with Raman spectroscopic monitoring.     

Changes in nail keratin observed by Raman spectroscopy after Nd:YAG laser treatment

Lasers and photodynamic therapy have been considered a convergence treatment for onychomycosis, which is a fungal infection on the nail bed and nail plate. Laser therapies have shown satisfactory results without significant complications for onychomycosis; however, the mechanism of clearing remains unknown. In this work, we investigated changes in the chemical structure of nail keratin induced by Nd:YAG laser using Raman spectroscopy. Toe nails with onychomycosis were treated with 1064 nm Nd:YAG laser. After laser treatment, the disulfide band (490–590 cm^?1) of nail keratin was rarely observed or was reduced in intensity. The amide I band (1500–1700 cm^?1) also showed changes induced by the laser. The α‐helical (1652 cm^?1) structures dominated the β‐sheet (1673 cm^?1) in nontreated nail, but the opposite phenomenon was observed after laser treatment.     

DISCRETE WAVELET TRANSFORM FOR DENOISING RAMAN SPECTRA OF HUMAN SKIN TISSUES USED IN A DISCRIMINANT DIAGNOSTIC ALGORITHM

In this work, we applied the discrete wavelet transform (DWT) method as a denoising tool for dispersive Raman spectra of skin samples, and we compared the results obtained with the low-order polynomial fitting in a discriminating model based on principal components analysis (PCA). We used a set of 50 Raman spectra of skin tissue fragments diagnosed as normal (N) (25 spectra) and basocellular cell carcinoma (BCC) (25 spectra). A denoising procedure using DWT and its inverse was employed, and the resulting spectra were compared to denoising using low-order polynomial fitting and adjacent averaging smoothing. The tissue spectral profile showed changes in the intensity of bands below 1400 cm^?1 for DWT compared to the denoising by polynomial and smoothing. By applying PCA and Mahalanobis distance in both groups processed, we verified that the filtering method does not alter significantly the discrimination of N and BCC tissues. However, the DWT denoising presented an interesting result, which showed the main components after decomposition of the Raman signal used in the reconstruction.     

Dental Enamel Caries (Early) Diagnosis and Mapping by Laser Raman Spectral Imaging

Abstract

A method for estimating the extent of tooth caries and providing imaging information based on Raman Spectral Imaging is suggested. This non‐destructive optical method is able to characterize and differentiate between normal enamel tooth surface, and initial and advanced tooth caries. Images and corresponding spectra were acquired from various tooth sites, and it was demonstrated that normal, white opaque, brown discoloured, and pitted tooth surfaces all have different distinct spectral features which characterize the different degrees of dental caries. Spectral analysis allows for detection of early changes in the surfaces of carious teeth, and the associated mapping capability allows for morphological characterization. It was found that the emission at 960 cm^?1, which corresponds to PO stretching in the hydroxyapatite bond, is the most significant and can be used for diagnosis of caries. The emissions at 1070 cm^?1 and at 590 cm^?1 can also be applied, but are less accurate. The results suggest that this technique may be further developed and applied for clinical diagnosis of initial and more advanced demineralization processes of the enamel tooth surfaces.     

Flexible and stable optical parametric oscillator based laser system for coherent anti‐Stokes Raman scattering microscopy

The characteristics of a stable and flexible laser system based on a synchronously pumped optical parametric oscillator (OPO) is presented. This OPO can offer very stable operation with both ～1 ps and ～300 fs outputs over a broad wavelength range, i.e., 920–1200 nm. Combining the pump tuning with the OPO tuning, a total Raman range of 1900–5500 cm^?1 is accessible. For maximum spectral sensitivity, the CARS microsope based on the ps laser system is presented in detail. The lateral resolution of the microscope is diffraction limited to be about 390 nm. Fast wavelength switching (sub‐second) between two Raman vibrational frequencies, i.e., 2848 cm^?1 for C? H aliphatic vibrations and 3035 cm^?1 for C? H aromatic vibrations is presented as an example, although this also extends to other Raman frequencies. The possibility of obtaining a multimodal imaging system based on the fs laser system is also discussed. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Label‐free monitoring of inflammatory tissue conditions using a carrageenan‐induced acute inflammation rat model

Although the confirmation of inflammatory changes within tissues at the onset of various diseases is critical for the early detection of disease and selection of appropriate treatment, most therapies are based on complex and time‐consuming diagnostic procedures. Raman spectroscopy has the ability to provide non‐invasive, real‐time, chemical bonding analysis through the inelastic scattering of photons. In this study, we evaluate the feasibility of Raman spectroscopy as a new, easy, fast, and accurate diagnostic method to support diagnostic decisions. The molecular changes in carrageenan‐induced acute inflammation rat tissues were assessed by Raman spectroscopy. Volumes of 0 (control), 100, 150, and 200 µL of 1% carrageenan were administered to rat hind paws to control the degree of inflammation. The prominent peaks at [1,062, 1,131] cm^?1 and [2,847, 2,881] cm^?1 were selected as characteristic measurements corresponding to the C–C stretching vibrational modes and the symmetric and asymmetric C–H (CH₂) stretching vibrational modes, respectively. Principal component analysis of the inflammatory Raman spectra enabled graphical representation of the degree of inflammation through principal component loading profiles of inflammatory tissues on a two‐dimensional plot. Therefore, Raman spectroscopy with multivariate statistical analysis represents a promising method for detecting biomolecular responses based on different types of inflammatory tissues.     

USE OF DISPERSIVE RAMAN SPECTROSCOPY IN THE DETERMINATION OF UNSATURATED FAT IN COMMERCIAL EDIBLE OIL- AND FAT-CONTAINING INDUSTRIALIZED FOODS

The present study aims at the use of Raman spectroscopy in the quantification of unsaturated fats in fat-containing foods, compared to the information available in the Nutritional Table, to obtain a non-destructive optical quantification of unsaturation. Raman spectra of edible oil, margarine, mayonnaise, hydrogenated fat, and butter were obtained with a near-infrared Raman spectrometer (830 nm). By analyzing selected bands in the regions of 1750, 1660, 1440, 1300, and 1260 cm^?1, the amount of total and unsaturated fat of samples of oil, margarine, and mayonnaise were correlated with the information displayed in the Nutritional Table. The amount of unsaturated trans fat in selected samples was correlated to the Raman shift of 1660 cm^?1. Dispersive Raman spectroscopy was shown to be effective in quantifying the unsaturated fats in oil, margarine, and mayonnaise, and trans fat in hydrogenated oils and butter.     

Two‐frequency CARS imaging by switching fiber laser excitation

To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber‐laser source. Stokes pulses were generated by soliton self‐frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid‐crystal retarder. Proof‐of‐principle is demonstrated by coherent anti‐Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm^?1, where polystyrene has a Raman transition, and 2,950 cm^?1, where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes.     

Quantification of anhydrous ethanol and detection of adulterants in commercial Brazilian gasoline by Raman spectroscopy

Raman spectroscopy can be used to evaluate the quality of fuels in a remote, rapid, and nondestructive manner without the need for reagents. In this study, Raman was used to quantify anhydrous ethanol in commercial gasoline and to detect peaks due to compounds commonly used for the adulteration of commercial gasoline. Samples of commercial gasoline were collected from fuel stations in the region of Santos, SP, Brazil. Samples of naphtha from the refinery, pure ethanol, and ethanol diluted in distilled water at concentrations close to the range used in the gasoline were also obtained and characterized. Raman spectra were collected using a dispersive Raman spectrometer (830?nm, 2?cm^?1 resolution in the 400–1800?cm^?1 spectral range). As expected, the spectra of commercial gasoline showed pronounced peaks of naphtha and ethanol. By using the peak intensities of the ethanol diluted in water, the ethanol concentration was found to be in the range of 27%?±?1% in most of the samples; some samples presented ethanol concentrations as high as 28.8%, suggesting adulteration. Some samples presented peaks at 766, 798, and 995?cm^?1 with higher intensities, suggesting the presence of an adulterant with organic characteristics, such as solvents with aromatic rings. Raman spectroscopy has been shown to be effective in determining the adulteration of commercial gasoline, which may contribute to rapid quality control of fuels at the point of sale.     

紫外-可见光双通道拉曼光谱仪光学设计

紫外拉曼光谱技术具有高强度拉曼散射、无荧光干扰的特点;可见光拉曼光谱技术可实现低波数、高分辨率探测。为兼具两种激发波长的优势,设计了一款对称分布的双Czerny-Turner光路聚焦于一个探测器的双通道拉曼光谱仪。通过元器件的选型和初始结构的计算,在不增加多余元器件的情况下,对弧矢方向像散进行补偿,避免了像面上的能量损失。配合Zemax软件对双通道光谱分别进行建模优化,最终实现了对400~5000 cm^-1（266 nm激发）和50~3500 cm^-1（633 nm激发）两段光谱的同时探测。均方根半径、点列图和调制传递函数等评价指标有效验证了设计的合理性和可行性。结果表明,两套拉曼光谱仪分别可达8 cm^-1和5 cm^-1分辨率,本设计具有高分辨率、低波数、多波长激发、集成化等优势。     

Near-field and confocal surface-enhanced resonance Raman spectroscopy at cryogenic temperatures

For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near‐field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano‐particles or nano‐clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm^?1 and 3000 cm^?1 at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.     

Near Infrared Raman Spectroscopy System for Real Time Monitoring of Fast Processes: A Resin Composite Photopolymerization Application

Abstract

This work describes the implementation of a technique for data monitoring using dispersive near‐infrared Raman spectroscopy that presents the advantage of non‐invasively being capable to make real time fast measurements. The degree of conversion of an odontological resin composite (A3, Z100^®, 3 M), photopolymerized by an LED (450 nm, 230 mW), was measured as an application of the technique. The Raman system consisted of a 785 nm Ti:Sapphire laser, a Chromex spectrograph with an entrance slit of 200 µm, and a liquid nitrogen cooled CCD detector. The system allowed acquisition of Raman spectra at times as short as 6 s. The progress of the photopolymerization was followed by obtaining Raman spectra every 6 s during 84 s. The intensity of the 1640 cm^?1 Raman peak, attributed to the aliphatic carbon double bonds, was chosen to calculate the conversion degree of the resin photopolymerization. Results showed that the degree of conversion presented an exponential growth, reaching a maximum of 50% after 60 s. Findings seem to confirm the trustworthiness of the system.     

Suppression of spurious background in low-frequency Raman spectroscopy

A monochromator capable of suppressing spurious laser radiation to a level required for low-frequency (<100 cm^?1) Raman spectroscopy is proposed. It has a high spectral resolution, contains a small number of optical elements, and can be easily included into the experimental optical scheme. The effect of using this monochromator in low-frequency Raman spectroscopy is illustrated by examples with test samples.     

Temperature measurement from the intensity ratio of the Raman-scattering lines in carbon tetrachloride constituting the liquid core of an optical fiber

A liquid-core optical fiber is used to measure the temperature from the intensity ratio of Ramanscattering lines. CCl₄ doped with benzene, which ensures conditions for the total internal reflection, serves as the fiber core. Measurements were performed at three Raman-scattering lines in CCl₄: 218, 314, and 459 cm^?1. The results obtained at 459 cm^?1 best correspond to the actual temperature. The advantages of using a liquidcore optical fiber and factors affecting the measurement results are discussed.     

Calcification of senile cataractous lens determined by Fourier transform infrared (FTIR) and Raman microspectroscopies

A calcified plaque on the surface of a senile cataractous lens (CL) isolated from a 79-year-old male patient was identified and its chemical composition quantified using Fourier transform infrared (FTIR) and confocal Raman microspectroscopies. The noncalcified area of the same CL and hydroxyapatite (HA) were selected as a control. Several unique absorption bands, at 960, 1034 and 1090 cm⁻¹ assigned to the ν₁ and ν₃ stretching modes of phosphate and at 875 cm⁻¹ attributed to carbonate, were clearly displayed in the infrared (IR) spectra of calcified plaque and HA. A peak at 961 cm⁻¹ due to the ν₁ stretching mode of phosphate was also evidenced in the Raman spectra of calcified plaque and HA. The calcified plaque formed within the lens protein was found to mainly consist of a mature HA, in which type-A carbonate apatites (11.4%), type-B carbonate apatites (55.6%) and liable surface carbonate ions (33.0%) were presented. A higher content of the liable carbonate implies that the calcification or mineralization in this calcified lens was incomplete and still in progress. Moreover, calcification seems not to influence the secondary structure of lens protein because both IR and Raman spectra for the lens protein in the noncalcified area and calcified plaque were similar. The result suggests that both microscopic FTIR and Raman spectroscopies were easy to perform and capable of determination of the chemical composition of a calcified CL.     

Identification and quantification of β-caryophyllene in copaiba oil using Raman spectroscopy

Copaiba oleoresin presents several compounds with known biologic activity and physiologic effects, including analgesic and insecticide properties. Among them are the terpenoids (mainly diterpenes and sesquiterpenes) with β-caryophyllene, the main representative of the terpenoids and considered to be a chemical marker. This study employed Raman spectroscopy and principal component analysis (PCA) techniques to identify and quantify the β-caryophyllene marker in copaiba oil samples purchased from popular markets in Brazil. A dispersive Raman spectrometer (830?nm, 250?mW, 2?cm^?1 spectral resolution) was used. Results showed the identification of the main Raman peaks from the β-caryophyllene in copaiba oil samples (main peaks at 507, 771, 1442, 1638, and 1673?cm^?1). The loading vector 2 (PC2) extracted the spectral information from β-caryophyllene in the samples and the eigenvalue 2 (score 2) allowed the estimation of the concentration of this marker in commercial samples, with the concentrations from 15 to 34%. Raman spectroscopy combined with PCA may be considered to be a potential analytical tool for the quality control of Copaifera oil samples by quantifying β-caryophyllene using its unique spectral information.     

Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

A neutron semiconductor detector based on TIInSe<Subscript>2</Subscript>

A new semiconductor detector of neutron radiation based on a TIInSe₂ crystal has been investigated. The detector is produced from a homogeneous semiconductor sample with two electric contacts and operates in an integrating mode. It is shown that, owing to its high sensitivity (～10^?13 A/(neutron cm^?2 s^?1)) and small size (the volume of the sensitive crystal element is ～7 mm³), the detector is capable of monitoring spatial, time, and intensity distributions of γ rays and neutrons in pulse research reactors.