KEYWORDS: Raman spectroscopy, Skin, Proteins, Tissues, In vivo imaging, Biological research, Melanoma, Chemical analysis, Collagen, Analytical research
The development of the disease leads to changes in the biochemical composition of biological tissues. Therefore, determination of the composition is important for medical diagnostics. In recent years, Raman spectroscopy has been used to study biological tissues. However, Raman spectra of most tissue components overlap significantly, and it is difficult to separate individual components. The aim of our study is to investigate the possibilities of the multivariate curve resolution alternating least squares method for the analysis of in vivo Raman spectra. We used a portable conventional spectroscopy setup. The analysis of Raman spectra of normal skin, keratosis, basal cell carcinoma, malignant melanoma and pigmented nevus was performed. As a result, we obtained spectral profiles corresponding to the contribution of the optical system and skin components: melanin, proteins, lipids, water, etc. The classification of the Raman spectra of various diseases (malignant vs. benign neoplasms, malignant melanoma vs pigmented neoplasms) by the contribution of the spectra of the components shows the classification accuracy about 70%. The obtained results show the possibility of unmixing several spectrally similar components using the multivariate curve resolution alternating least squares analysis even under noisy conditions of the recorded Raman spectra. The method may be used for the analysis of Raman spectra with a low signal-to-noise ratio.
In this study in vivo optical diagnostic of skin cancer was performed using autofluorescence spectroscopy in the near infrared region to detect amelanotic melanoma among other skin cancer types.
This paper presents the application of the optical methods of Raman spectroscopy and autofluorescence analysis for studying of the skin diagnostic features. Benign and malignant skin tumors from 89 patients were measured using portable spectroscopic system with near-infrared 785 nm laser excitation. Classifications of the different skin tumors were made using partial least squares methods with discriminant analysis in two tasks: melanoma from other malignant skin tumors (basal cell carcinoma and squamous cell carcinoma) with 0.95 sensitivity and 0.86 specificity and melanoma from pigmented nevus with 0.68 sensitivity and 0.78 specificity. The main findings establish that combined analysis of the autofluorescence and Raman spectral signatures of the different skin tumors are more effective in comparison with using of the only Raman spectroscopy. Also, results of classifications show the tumor spectra contain most useful information rather than tumor spectra normalized to the corresponding normal skin spectra.
Changes of concentrations of plasma free amino acids (PFAAs) is an essential feature of protein metabolic abnormalities in cancer patients. In this study, we aim to decompose Raman spectra of mixtures of amino acids. The effect of noise on the decomposition result is investigated. The experimentally measured spectra of amino acids and artificially simulated spectra of their mixtures are studied. As a decomposition method, Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) analysis is used. It is shown that one can evaluate the concentration of amino acids in the mixtures using the Raman spectra of the mixtures and the spectra of pure amino acids. The results can be used in further research on lung cancer.
We investigated pigmented skin tumour lesions in vivo and ex vivo, including benign and dysplastic nevi, as well as malignant lesions, such as pigmented basal cell carcinoma (BCC) and malignant melanoma (MM) lesions, to obtain a complex view about the feasibility of different excitation sources solely and/or in combination to induce fluorescence signal useful for diagnosis of various low-fluorescent cutaneous neoplasia. A specialized multispectral analysis of the data obtained was applied by using excitation in broad spectral range, covering ultraviolet, visible and near-infrared spectral range, that contribute considerably to: (1) fundamental determination of tumour tissues’ spectral properties, and (2) to increase the accuracy in determining the type of cutaneous pathology. The chromophores, related to the formation of ultraviolet and visible (UV-VIS) fluorescence in human normal skin and its pigmented lesions are mainly amino acids – tryptophan, tyrosine; structural proteins and their cross-links – collagen, elastin, keratin; co-enzymes - NADH, flavins; vitamins and lipids. In the near-infrared (NIR) spectral region, skin fluorescence emission properties are related to the presence of melanin pigment, lipids and endogenous porphyrins, if any, as the highest impact on the resultant emission spectrum is due to the melanin compound.
A comparative in vivo study of skin tumors and normal skin was performed using Raman and autofluorescence spectral signals simultaneously recorded in the range of 800–1000 nm with a help of portable spectrometer and 785 nm excitation laser. The 1012 spectra were collected from 506 patients of the Samara Clinical Oncological Dispensary. The classification of spectral data for various tumors and normal skin was carried out by the PLS-DA method. The bands with most important spectral differences between analyzed skin tissues were found with the projection on latent structures analysis. The spectral differences showed significant changes among normal skin and tumors associated with such biochemicals as elastin, collagen, melanin, nucleic acid, lipids, phenylalanine. Results of the analysis were presented by utilizing sensitivity, specificity and accuracy and receiving operating characteristic (ROC) curve. To classify 314 spectra of benign tumors and 314 spectra of normal skin the mean accuracy was found to be a 0.86; for classification of 192 spectra of skin cancer and 192 spectra of normal skin the mean accuracy was found to be a 0.84; when the benign tumors and skin cancer were combined and differentiated from all normal skin 0.79 mean accuracy was obtained. The achieved results demonstrates Raman and autofluorescence spectra efficiency in highlighting biochemical changes during tumor growth.
In the paper, we introduce an additive simulation approach of Raman light scattering by skin cancer using the Monte Carlo method. Raman light scattering from normal skin and malignant melanoma is investigated. A two-stage algorithm for simulating Raman light scattering from skin based on the known photon transport algorithm has been developed. A method for additive modeling of skin pathologies is proposed. The main idea of this method is a hypothesis that an experimental Raman spectrum of normal skin, obtained by averaging in vivo Raman spectra of normal skin, may be served as a “substrate” for the feature simulated Raman spectrum. Thus, the pathology, for their part, may be “grown” by adding on this “substrate” Raman specific components set related to a tumor type. Additive simulation of malignant melanoma has been carried out. The possibility of using the developed algorithm to determine the component composition of the skin by the in vivo Raman spectrum of skin is discussed. An attempt to evaluate the change in the concentration of skin components during the development of cancer has been made.
We demonstrate the applicability of near-infrared (NIR) autofluorescence (AF) of skin tissues to differentiating neoplasms based on performing a series of experiments with in vivo and ex vivo skin tumors and analyzing the skin AF spectral shape, the excitation–emission matrices, and the photobleaching properties of malignant and benign neoplasms. The melanin-pigmented lesions showed an increase of the AF in comparison to nonpigmented tissue fluorescent emission using excitation at 785 nm. Autofluorescent spectral differences can be associated with different concentration of melanocytes cells in the investigated skin lesions. The differences in excitation–emission matrices for tested tissues prove that melanin is the dominant NIR fluorophore in human skin. Further, we found that the photobleaching properties of normal skin and neoplasms differ significantly. The highlighted differences in skin tissue AF response can be used in rapid analysis of large tissue areas and can complement other methods of skin tumor detection.
In this in vivo pilot clinical trial, an estimation of NIR spectral features of malignant and benign tumors and normal skin was carried out. The human tissue spectra simultaneously including Raman spectroscopy and autofluorescence signals were registered using the portable spectroscopic equipment with 785 nm excitation laser and spectra registration in the 800-950 nm area. Spectral data were analyzed using PLS-DA method extracted the relevant information that allow for classification of skin neoplasm type. The most important spectral bands were found with the variable importance in projection analysis. We analyzed four models: basal cell carcinoma versus normal skin, basal cell carcinoma versus benign tumors, basal cell carcinoma versus melanoma and malignant tumors versus normal skin and benign neoplasms. The results of the neoplasm groups differentiating for each model were presented by using boxplot diagrams and receiving operating characteristic (ROC) curve approach. Achieved results demonstrate high accuracy of Raman spectroscopy and autofluorescence combination in NIR for the classification of malignant and benign skin lesions.
The state of internal human homeostasis, namely the function of the internal organs - the endocrine system, the digestive tract, the nervous, hematopoietic, cardiovascular and other systems, is closely related to the skin condition. Changes in the skin biochemistry are a reflection of the internal state of the human body. Therefore, the analysis of changes in the composition of human skin various layers is one of complex parts of therapeutic disciplines. In addition to the laboratory analysis methods used today, a variety of physical methods can be successfully used to study the component composition of the human skin. Methods of Raman Spectroscopy and autofluorescence analysis can detect changes in the component composition of the skin at the molecular level. In current study we used Raman spectroscopy and autofluorescence analysis in visible and NIR regions for the analysis of human skin spectral characteristics in the presence of various influencing factors including chronic kidney transplant dysfunction.
The Raman and autofluorescence spectral characteristics of studied samples in NIR region were registered using the experimental setup, incorporated a high-resolution spectrometer with integrated cooled digital camera, a fiber-optic Raman probe and the laser module with central wavelength 785 nm. The autofluorescence human skin response in visible region was registered by portable diagnostic fluorimeter, which provide an excitation light source across the 350-400 nm range and measured light intensity within the 420-600 nm range. In this study we describe the design and results of the tests on volunteers of portable fluorescence meter based on two photodiodes. One channel of such fluorometer is used for measurement of autofluorescence intensity, another one - for intensity of elastically scattered radiation, which can be used as reference. The processing of experimental data was performed on the basis of regression analysis.
We performed the comparative research of Raman experimental data and visible autofluorescence analysis results. We estimated correlations between Raman and autofluorescence signals and also find informative Raman bands that may be used as predictors of general condition of the body. These bands lie in 1170 – 1700 cm-1 region. We demonstrated the possibility to measure melanin and lipofuscin levels in the skin, as they are the hallmarks of skin aging; and demonstrated the possibility to measure a level of advanced glycation end products in the skin, advanced glycation end products as and lipofuscins are markers of general body condition. In addition, we have found informative spectral bands characterizing changes in the component composition of the skin in the presence of various influencing factors as kidney diseases.
The differentiation of skin melanomas and basal cell carcinomas (BCCs) was demonstrated based on combined analysis of Raman and autofluorescence spectra stimulated by visible and NIR lasers. It was ex vivo tested on 39 melanomas and 40 BCCs. Six spectroscopic criteria utilizing information about alteration of melanin, porphyrins, flavins, lipids, and collagen content in tumor with a comparison to healthy skin were proposed. The measured correlation between the proposed criteria makes it possible to define weakly correlated criteria groups for discriminant analysis and principal components analysis application. It was shown that the accuracy of cancerous tissues classification reaches 97.3% for a combined 6-criteria multimodal algorithm, while the accuracy determined separately for each modality does not exceed 79%. The combined 6-D method is a rapid and reliable tool for malignant skin detection and classification.
This work is devoted to study the possibility of plasma proteins (albumin, globulins) concentration measurement using Raman spectroscopy setup. The blood plasma and whole blood were studied in this research. The obtained Raman spectra showed significant variation of intensities of certain spectral bands 940, 1005, 1330, 1450 and 1650 cm-1 for different protein fractions. Partial least squares regression analysis was used for determination of correlation coefficients. We have shown that the proposed method represents the structure and biochemical composition of major blood proteins.
In this study we demonstrate a comparative analysis of blood serum and normal human skin by Raman spectroscopy with application of different spectroscopic equipment. For serum analysis we measure a total concentration of proteins and compared it with intensity of 1002 cm-1 Raman peak. Standard deviation for protein control in blood serum differed from 7.4% to 19% for different spectroscopic setups. For human skin control we used three Raman peaks near 1340, 1450 and 1650 cm-1. Measurements of different skin samples were analyzed on the phase plane to find areas corresponding to the normal skin. Taking into account the different sensitivities of the detected signal with different detectors in the spectral range 810-950 nm we calculated correction coefficients allowed for making comparison of spectral measurements made on different spectrometers with ranging not exceeding 21%.
Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.
The combined application of Raman and autofluorescence spectroscopy in visible and near infrared regions for the analysis of malignant neoplasms of human skin was demonstrated. Ex vivo experiments were performed for 130 skin tissue samples: 28 malignant melanomas, 19 basal cell carcinomas, 15 benign tumors, 9 nevi and 59 normal tissues. Proposed method of Raman spectra analysis allows for malignant melanoma differentiating from other skin tissues with accuracy of 84% (sensitivity of 97%, specificity of 72%). Autofluorescence analysis in near infrared and visible regions helped us to increase the diagnostic accuracy by 5-10%. Registration of autofluorescence in near infrared region is realized in one optical unit with Raman spectroscopy. Thus, the proposed method of combined skin tissues study makes possible simultaneous large skin area study with autofluorescence spectra analysis and precise neoplasm type determination with Raman spectroscopy.
The fluorescence and Raman spectroscopy (RS) combined method of in vivo detection of malignant human skin cancer
was demonstrated. The fluorescence analysis was used for detection of abnormalities during fast scanning of large tissue
areas. In suspected cases of malignancy the Raman spectrum analysis of biological tissue was performed to determine
the type of neoplasm. A special RS phase method was proposed for in vivo identification of skin tumor. Quadratic
Discriminant Analysis was used for tumor type classification on phase planes. It was shown that the application of phase
method provides a diagnosis of malignant melanoma with a sensitivity of 89% and a specificity of 87%.
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