Noninvasive assessments of skin glycated proteins by fluorescence and Raman techniques in diabetics and nondiabetics

Diabetes is a complex metabolic disease and has chronic complications. It has been considered a serious public health problem. The aim of the current study was to evaluate skin glycated proteins through fluorescence and Raman techniques. One hundred subjects were invited to participate in the study. Six volunteers did not attend due to exclusion criteria or a change of mind about participating. Therefore, 94 volunteers were grouped according to age range (20‐80 years), health condition (nondiabetic, with insulin resistance [IR] and/or diabetic) and Fitzpatrick skin type (I‐VI). The fluorescence spectrometer and the portable Raman spectroscopy system were used to measure glycated proteins from the skin. There was elevated skin autofluorescence in healthy middle‐aged and elderly subjects, as well as in patients with IR and/or diabetes. Regarding Raman spectroscopy, changes in the skin hydration state, degradation of type I collagen and greater glycation were related for diabetes and chronological aging. Weak and positive correlation between the skin autofluorescence and the Raman peaks ratio (855/876) related to the glycated proteins was also found. Raman spectroscopy shows several bands for spectral analyses, complementing the fluorescence data. Therefore, this study contributes to understanding of the optical of human skin for noninvasive diabetes screening.      

Real‐time endoscopic Raman spectroscopy for in vivo early lung cancer detection

Currently the most sensitive method for localizing lung cancers in central airways is autofluorescence bronchoscopy (AFB) in combination with white light bronchoscopy (WLB). The diagnostic accuracy of WLB + AFB for high grade dysplasia (HGD) and carcinoma in situ is variable depending on physician's experience. When WLB + AFB are operated at high diagnostic sensitivity, the associated diagnostic specificity is low. Raman spectroscopy probes molecular vibrations and gives highly specific, fingerprint‐like spectral features and has high accuracy for tissue pathology classification. In this study we present the use of a real‐time endoscopy Raman spectroscopy system to improve the specificity. A spectrum is acquired within 1 second and clinical data are obtained from 280 tissue sites (72 HGDs/malignant lesions, 208 benign lesions/normal sites) in 80 patients. Using multivariate analyses and waveband selection methods on the Raman spectra, we have demonstrated that HGD and malignant lung lesions can be detected with high sensitivity (90%) and good specificity (65%).

     

Identification and characterization of bladder cancer by low‐resolution fiber‐optic Raman spectroscopy

Raman spectroscopy has been proved to be a promising diagnostic technique for various cancers detection. A major drawback for its clinical translation is the intrinsic weakness of Raman effects. Highly sensitive equipment and optimal measurement conditions are generally applied to overcome this drawback. However, these equipment are usually bulky, expensive and may also be easily influenced by surrounding environment. In this preliminary work, a low‐resolution fiber‐optic Raman sensing system is applied to evaluate the diagnostic potential of Raman spectroscopy to identify different bladder pathologies ex vivo. A total number of 262 spectra taken from 32 bladder specimens are included in this study. These spectra are categorized into 3 groups by histopathological analysis, namely normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors. Principal component analysis fed artificial neural network are used to train a classification model for the spectral data with 10‐fold cross‐validation and an overall prediction accuracy of 93.1% is obtained. The sensitivities and specificities for normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors are 88.5% and 95.1%, 90.3% and 98%, and 97.5% and 96.4%, respectively. These results demonstrate the potential of using a low‐resolution fiber‐optic Raman system for in vivo bladder cancer diagnosis.      

Chemico‐mechanical imaging of Barrett's oesophagus

Barrett's oesophagus is a condition characterized by a change in the lining of the oesophagus that markedly increases the risk of adenocarcinoma. We demonstrate the first site‐matched application of Brillouin microscopy, Raman microscopy and FTIR micro‐spectroscopic imaging to ex‐vivo epithelial tissue – Barrett's oesophagus. The mechanical and chemical characters of the epithelium were assessed in histological sections from a patient subjected to endoscopic oesophageal biopsy. Previous studies have shown that both these properties change within the oesophageal wall, owing to the presence of distinct cellular and extracellular constituents which are putatively affected by oesophageal cancer. Brillouin microscopy enables maps of elasticity of the epithelium to be obtained, whilst Raman and FTIR imaging provide ’chemical images' without the need for labelling or staining. This site‐matched approach provides a valuable platform for investigating the structure, biomechanics and composition of complex heterogeneous systems. A combined Brillouin‐Raman device has potential for in‐vivo diagnosis of pathology.

First application of site‐matched micro Brillouin, Raman and FTIR spectroscopic imaging to epithelial tissue in Barrett's oesophagus     

Quantitative and rapid estimations of human sub‐surface skin mass using ultra‐high‐resolution spectral domain optical coherence tomography

Non‐invasive and quantitative estimations for the delineation of sub‐surface tumor margins could greatly aid in the early detection and monitoring of the morphological appearances of tumor growth, ensure complete tumor excision without the unnecessary sacrifice of healthy tissue, and facilitate post‐operative follow‐up for recurrence. In this study, a high‐speed, non‐invasive, and ultra‐high‐resolution spectral domain optical coherence tomography (UHR‐SDOCT) imaging platform was developed for the quantitative measurement of human sub‐surface skin mass. With a proposed robust, semi‐automatic analysis, the system can rapidly quantify lesion area and shape regularity by an en‐face‐oriented algorithm. Various sizes of nylon sutures embedded in pork skin were used first as a phantom to verify the accuracy of our algorithm, and then in vivo, feasibility was proven using benign human angiomas and pigmented nevi. Clinically, this is the first step towards an automated skin lesion measurement system.

In vivo optical coherence tomography (OCT) image of angioma (A). Thin red arrows point to a blood vessel (BV).     

Real‐time in vivo cancer diagnosis using raman spectroscopy

Raman spectroscopy has becoming a practical tool for rapid in vivo tissue diagnosis. This paper provides an overview on the latest development of real‐time in vivo Raman systems for cancer detection. Instrumentation, data handling, as well as oncology applications of Raman techniques were covered. Optic fiber probes designs for Raman spectroscopy were discussed. Spectral data pre‐processing, feature extraction, and classification between normal/benign and malignant tissues were surveyed. Applications of Raman techniques for clinical diagnosis for different types of cancers, including skin cancer, lung cancer, stomach cancer, oesophageal cancer, colorectal cancer, cervical cancer, and breast cancer, were summarized.

Schematic of a real‐time Raman spectrometer for skin cancer detection. Without correction, the image captured on CCD camera for a straight entrance slit has a curvature. By arranging the optic fiber array in reverse orientation, the curvature could be effectively corrected.     

Confocal Raman spectroscopic imaging for in vitro monitoring of active ingredient penetration and distribution in reconstructed human epidermis model

Topically applied active cosmetic ingredients (ACI) or active pharmaceutical ingredients (API) efficacy is directly related to their efficiency of penetration in the skin. In vitro reconstructed human epidermis surrogate models offer in vivo like skin samples for transdermal studies. Using Delipidol®, an ACI currently used in the cosmetics industry, the capabilities to deliver accurate distribution maps and penetration profiles of this molecule by means of confocal Raman spectroscopic imaging have been demonstrated. Using a non‐negative constrained least squares (NCLS) approach, contribution of specific molecules can be estimated at each point of spectral maps in order to deliver semi‐quantitative heat maps representing the ACI levels in the different skin layers. The concentration profiles obtained are approximately single exponential for all 3 time points evaluated, with a consistent decay constant, which is independent of the sublayer structure. Notably, however, there is no significant penetration into the lower basal layers until a critical concentration is built up, after 3 hours. Combination of Raman confocal imaging with spectral unmixing methods such as NCLS is demonstrated to be a relevant approach for in vitro biological evaluation of cosmetic and pharmaceutical active ingredients and could easily be implemented as a screening tool for industrial use.      

Raman spectroscopy of blood serum for Alzheimer's disease diagnostics: specificity relative to other types of dementia

The key moment for efficiently and accurately diagnosing dementia occurs during the early stages. This is particularly true for Alzheimer's disease (AD). In this proof‐of‐concept study, we applied near infrared (NIR) Raman microspectroscopy of blood serum together with advanced multivariate statistics for the selective identification of AD. We analyzed data from 20 AD patients, 18 patients with other neurodegenerative dementias (OD) and 10 healthy control (HC) subjects. NIR Raman microspectroscopy differentiated patients with more than 95% sensitivity and specificity. We demonstrated the high discriminative power of artificial neural network (ANN) classification models, thus revealing the high potential of this developed methodology for the differential diagnosis of AD. Raman spectroscopic, blood‐based tests may aid clinical assessments for the effective and accurate differential diagnosis of AD, decrease the labor, time and cost of diagnosis, and be useful for screening patient populations for AD development and progression.

Multivariate data analysis of blood serum Raman spectra allows for the differentiation between patients with Alzheimer's disease, other types of dementia and healthy individuals.     

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

We present results of Raman spectroscopic studies carried out on optically trapped red blood cells with Raman excitation wavelength in Q‐band region of the hemoglobin (Hb) absorption spectrum. The results obtained suggest that when exposed to the Raman excitation laser the RBCs get deoxygenated due to photo‐dissociation of oxygen from hemoglobin. For smaller exposure durations (5 s) the level of deoxygenation increases with an increase in power. However, for longer exposure durations the deoxygenated hemoglobin in the cells gets irreversibly oxidized to form a low spin ferric derivative of hemoglobin. The rate of oxidation depends upon the initial level of deoxygenation; higher the initial level of deoxygenation, higher is the rate of oxidation. However, the RBCs deoxygenated via oxygen deprivation (i.e. N₂ purging) were found to be very stable against any laser induced effect. These observations suggests that in case of laser induced deoxygenation of RBCs the free oxygen generated by photo‐dissociation acts as the oxidizing agent and leads to oxidative damage of the RBCs.

     

Simultaneous fingerprint and high‐wavenumber fiber‐optic Raman spectroscopy enhances real‐time in vivo diagnosis of adenomatous polyps during colonoscopy

Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm^–1) and HW (i.e., 2800–3600 cm^–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.

     

In vivo detection of human cutaneous beta‐carotene using computational optical clearing

The content of dermal beta‐carotene can be a good indicator showing the body health. Because, it is involved in production of vitamin A maintaining healthy skin and mucous membranes. Also, it reduces the risk of cardiovascular diseases and its antioxidant capacity prevents the formation of cancerous cells. In this work, we use Raman spectroscopy and a low‐cost diffuse reflectance spectroscopy (DRS) to detect the dermal beta‐carotene spectra. We apply computational optical clearing (OC) method to in vivo evaluation the concentration of this chromophore. The results show that Raman spectroscopy is a good tool for in vitro detection of carotenoids but is not able to clearly discriminate the individual carotenoids in skin tissue in vivo. The results also show that using OC enhances the ability of low‐cost diffuse reflection spectroscopy for in vivo detection of dermal beta‐carotene in humans. This method can be used as a low‐cost and portable device to screening the concentration of chromophores such as melanin and carotenoid molecules for oncological studies.     

Combining near-infrared-excited autofluorescence and Raman spectroscopy improves in vivo diagnosis of gastric cancer

This study aims to evaluate the diagnostic utility of the combined near-infrared (NIR) autofluorescence (AF) and Raman spectroscopy for improving in vivo detection of gastric cancer at clinical gastroscopy. A rapid Raman endoscopic technique was employed for in vivo spectroscopic measurements of normal (n=1098) and cancer (n=140) gastric tissues from 81 gastric patients. The composite NIR AF and Raman spectra in the range of 800-1800 cm(-1) were analyzed using principal component analysis (PCA) and linear discriminant (LDA) to extract diagnostic information associated with distinctive spectroscopic processes of gastric malignancies. High quality in vivo composite NIR AF and Raman spectra can routinely be acquired from the gastric within 0.5s. The integrated intensity over the range of 800-1800 cm(-1) established the diagnostic implications (p=1.6E-14) of the change of NIR AF intensity associated with neoplastic transformation. PCA-LDA diagnostic modeling on the in vivo tissue NIR AF and Raman spectra acquired yielded a diagnostic accuracy of 92.2% (sensitivity of 97.9% and specificity of 91.5%) for identifying gastric cancer from normal tissue. The integration area under the receiver operating characteristic (ROC) curve using the combined NIR AF and Raman spectroscopy was 0.985, which is superior to either the Raman spectroscopy or NIR AF spectroscopy alone. This work demonstrates that the complementary Raman and NIR AF spectroscopy techniques can be integrated together for improving the in vivo diagnosis and detection of gastric cancer at endoscopy.     

Serum calcitonin negative Medullary thyroid carcinoma

Background

Epithelial ovarian neoplasms are an important cause of morbidity and mortality in women. The surgical management of ovarian neoplasms depends on their correct categorization as benign, borderline or malignant. This study was undertaken to evaluate the accuracy of intra-operative frozen section in the diagnosis of various categories of ovarian neoplasms.

Methods

Intraoperative frozen section diagnosis was retrospectively evaluated in 217 patients with suspected ovarian neoplasms who underwent surgery as primary line of therapy at our institution. This was compared with the final histopathologic diagnosis on paraffin sections.

Results

In 7 patients (3.2%) no opinion on frozen section was possible. In the remaining 210 patients frozen section report had a sensitivity of 100%, 93.5% and 45.5% for benign, malignant and borderline tumors. The corresponding specificities were 93.2%, 98.3% and 98.5% respectively. The overall accuracy of frozen section diagnosis was 91.2%. The majority of cases of disagreement were in the mucinous and borderline tumors.

Conclusion

Intraoperative frozen section has high accuracy in the diagnosis of suspected ovarian neoplasms. It is a valuable tool to guide the surgical management of these patients and should be routinely used in all major oncology centers.     

Effect of growth media and phase on Raman spectra and discrimination of mycobacteria

When developing a Raman spectral library to identify bacteria, differences between laboratory and real world conditions must be considered. For example, culturing bacteria in laboratory settings is performed under conditions for ideal bacteria growth. In contrast, culture conditions in the human body may differ and may not support optimized bacterial growth. To address these differences, researchers have studied the effect of conditions such as growth media and phase on Raman spectra. However, the majority of these studies focused on Gram‐positive or Gram‐negative bacteria. This article focuses on the influence of growth media and phase on Raman spectra and discrimination of mycobacteria, an acid‐fast genus. Results showed that spectral differences from growth phase and media can be distinguished by spectral observation and multivariate analysis. Results were comparable to those found for other types of bacteria, such as Gram‐positive and Gram‐negative. In addition, the influence of growth phase and media had a significant impact on machine learning models and their resulting classification accuracy. This study highlights the need for machine learning models and their associated spectral libraries to account for various growth parameters and stages to further the transition of Raman spectral analysis of bacteria from laboratory to clinical settings.      

Laser‐induced capillary leakage for blood biomarker detection and vaccine delivery via the skin

Circulation system is the center for coordination and communication of all organs in our body. Examination of any change in its analytes or delivery of therapeutic drugs into the system consists of important medical practice in today's medicine. Two recent studies prove that brief illumination of skin with a low powered laser, at wavelengths preferentially absorbed by hemoglobin, increases the amount of circulating biomarkers in the epidermis and upper dermis by more than 1,000‐fold. When probe‐coated microneedle arrays are applied into laser‐treated skin, plasma blood biomarkers can be reliably, accurately, and sufficiently quantified in 15～30 min assays, with a maximal detection in one hr in a manner independent of penetration depth or a molecular mass of the biomarker. Moreover, the laser treatment permits a high efficient delivery of radiation‐attenuated malarial sporozoites (RAS) into the circulation, leading to robust immunity against malaria infections, whereas similar immunization at sham‐treated skin elicits poor immune responses. Thus this technology can potentially instruct designs of small, portable devices for onsite, in mobile clinics, or at home for point‐of‐care diagnosis and drug/vaccine delivery via the skin.

Laser‐induced capillary leakage ( a ) to induce extravasation of circualing molecules only ( b ) or facilitate entry of attenuated malaria sporozoites into the capillary ( c ). Skin illumination with a laser preferably absorbed by hemoglobin causes dilation of the capillary beneath the skin. The extravasated molecules can be sufficiently measured in the skin or guide sporozoites to enter the vessel.     

Raman spectroscopic identification of Mycobacterium tuberculosis

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation‐independent Raman microspectroscopy emerges as a perfect tool for a rapid on‐the‐spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two‐level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first‐line diagnostic point‐of‐care tool for the confirmation of tuberculosis disease.

     

High sensitivity non‐invasive detection of calcifications deep inside biological tissue using Transmission Raman Spectroscopy

The aim of this research was to develop a novel approach to probe non‐invasively the composition of inorganic chemicals buried deep in large volume biological samples. The method is based on advanced Transmission Raman Spectroscopy (TRS) permitting chemical specific detection within a large sampling volume. The approach could be beneficial to chemical identification of the breast calcifications detected during mammographic X‐ray procedures. The chemical composition of a breast calcification reflects the pathology of the surrounding tissue, malignant or benign and potentially the grade of malignancy. However, this information is not available from mammography, leading to excisional biopsy and histopathological assessment for a definitive diagnosis. Here we present, for the first time, a design of a new high performance deep Raman instrument and demonstrate its capability to detect type II calcifications (calcium hydroxyapatite) at clinically relevant concentrations and depths of around 40 mm in phantom tissue. This is around double the penetration depth achieved with our previous instrument design and around two orders of magnitude higher than that possible when using conventional Raman spectroscopy.

     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Immunohistochemical profile of galectin-8 expression in benign and malignant tumors of epithelial, mesenchymatous and adipous origins, and of the nervous system

This study aims to investigate whether the immunohistochemical expression of galectin-8 could be used as a diagnostic marker in tumor tissues of various histogenetic origins including specimens from epithelial (n=145), mesenchymatous (n=16), adipous (n=10) and central and peripheral nervous system (n=25) tissue, and 4 mesotheliomas. Immunohistochemical reactions were carried out with a polyclonal anti-galectin-8 antibody and histological slides from tissues derived from the files of the Laboratory of Anatomopathology of University Erasmus Hospital, Brussels. Formalin-fixed paraffin-embedded tissues of 45 normal cases as well as 41 benign and 114 malignant tumors were studied. Marked decreases in immunohistochemical galectin-8 expression were observed in colon (p=0.001), pancreas (p=0.007), liver (p=0.0008), skin (p=0.002) and larynx (p=0.02) tissue when comparing malignant tissue to normal tissue and/or benign tumors. The reverse relationship was observed for breast tissue (p=0.007). No statistically significant differences (p>0.05) were detected when comparing normal tissue and/or benign to malignant tumors in lung, bladder, kidney, prostate and stomach tissue. Significant galectin-8 expression was also measured in non-epithelial tissue including tumors of the central and peripheral nervous system as well as in skeletal muscle and mesotheliomas. Immunohistochemical monitoring of galectin-8 thus reveals an organ-type-dependent regulation of expression upon malignant transformation of various tissue types of epithelial origin. This observation will prompt further studies to delineate any relationship with prognosis.     

Differential Gene Expression Analysis by DNA Microarray Technology and Its Application in Molecular Oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools aim to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the results of detailed combinatory analysis of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such genetic portraits allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the molecular nosology of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their application in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.