Mobile multi-configuration clinical translational Raman system for oral cancer application.

Early diagnosis of oral cancer is critical to improve the survival rate of patients. Raman spectroscopy, a non-invasive spectroscopic technique, has shown potential in identifying early-stage oral cancer biomarkers in the oral cavity environment. However, inherently weak signals necessitate highly sensitive detectors, which restricts widespread usage due to high setup costs. In this research, the fabrication and assembly of a customised Raman system that can adapt three different configurations for the in vivo and ex vivo analysis is reported. This novel design will help in reducing the cost required to have multiple Raman instruments specific for a given application. First, we demonstrated the capability of a customized microscope for acquiring Raman signals from a single cell with high signal-to-noise ratio. Generally, when working with liquid samples with low concentration of analytes (such as saliva) under a microscope, excitation light interacts with a small sample volume, which may not be representative of whole sample. To address this issue, we have designed a novel long-path transmission set-up, which was found to be sensitive towards low concentration of analytes in aqueous solution. We further demonstrated that the same Raman system can be incorporated with the multimodal fibre optical probe to collect in vivo data from oral tissues. In summary, this flexible, portable, multi-configuration Raman system has the potential to provide a cost-effective solution for complete screening of precancer oral lesions.

In vivo Raman spectroscopic characteristics of different sites of the oral mucosa in healthy volunteers.

OBJECTIVES: Investigate the biochemistry of in vivo healthy oral tissues through Raman spectroscopy. We aimed to characterize the biochemical features of healthy condition in oral subsites (buccal mucosa, lip, tongue, and gingiva) of healthy subjects. More specifically, we investigated Raman spectral characteristics and biochemical content of in vivo healthy tissues on Brazilian population. This characterization can be used to better define normal tissue and improve the detection of oral premalignant conditions in future studies. MATERIALS AND METHODS: For spectroscopic analysis a Raman spectrometer (Kaiser Optical Systems imaging spectrograph Holospec, f / 1.8i-NIR) coupled with a laser 785 nm, 60 mW was used. Raman measurements were obtained by means of an optical fiber (EMVision fiber optic probe) coupled between the laser and the spectrometer. Three spectra per site were acquired from the lip, buccal mucosa, tongue, and gingiva of ten healthy volunteers. This resulted in 30 spectra per oral sub-site and in total 120 spectra. RESULTS: We report detailed biochemical information on these subsites and their relative composition based on deconvolution studies of their spectra. Finally, we also report classification efficiency of 61, 83, 41, and 93% for buccal, gingiva, lip, and tongue respectively after applying multivariate statistical tools. CONCLUSIONS: We quantitated the contribution of various biochemicals in terms of percentage, and this will enable comparison not only across anatomical sites but also across studies. Raman spectroscopy can rapidly probe tissue biochemistry of healthy oral regions. Moreover, the study suggests the possibility of using Raman spectroscopy combined with signal processing and multivariate analysis methods to differentiate the oral sites in healthy conditions and compare with pathological conditions in future studies. CLINICAL RELEVANCE: The spectral characterization of the healthy condition of oral tissues by a noninvasive, label-free, and real-time analytical techniques is important to create a spectral reference for future diagnosis of pathological conditions.

Fluorescence lifetime evaluation of whole soils from the Amazon rainforest.

Time-resolved fluorescence spectroscopy (TRFS) is a new tool that can be used to investigate processes of interaction between metal ions and organic matter (OM) in soils, providing a specific analysis of the structure and dynamics of macromolecules. To the best of our knowledge, there are no studies in the literature reporting the use of this technique applied to whole/non-fractionated soil samples, making it a potential method for use in future studies. This work describes the use of TRFS to evaluate the fluorescence lifetimes of OM of whole soils from the Amazon region. Analysis was made of pellets of soils from an oxisol-spodosol system, collected in São Gabriel da Cachoeira (Amazonas, Brazil). The fluorescence lifetimes in the oxisol-spodosol system were attributed to two different fluorophores. One was related to complexation of an OM fraction with metals, resulting in a shorter fluorophore lifetime. A short fluorescence lifetime (2-12 ns) could be associated with simpler structures of the OM, while a long lifetime (19-66 ns) was associated with more complex OM structures. This new TRFS technique for analysis of the fluorescence lifetime in whole soil samples complies with the principles of green chemistry.

Infrared spectroscopy for fast screening of diabetes and periodontitis.

SIGNIFICANCE: FT-IR is an important and emerging tool, providing information related to the biochemical composition of biofluids. It is important to demonstrate that there is an efficacy in separating healthy and diseased groups, helping to establish FT-IR uses as fast screening tool. AIM: Via saliva diagnosis evaluate the accuracy of FT-IR associate with machine learning model for classification among healthy (control group), diabetic (D) and periodontitis (P) patients and the association of both diseases (DP). APPROACH: Eighty patients diagnosed with diabetes and periodontitis through conventional methods were recruited and allocated in one of the four groups. Saliva samples were collected from participants of each group (n = 20) and were processed using Bruker Alpha II spectrometer in a FT-IR spectral fingerprint region between 600 and-1800 cm-1, followed by data preprocessing and analysis using machine learning tools. RESULTS: Various FTI-R peaks were detectable and attributed to specific vibrational modes, which were classified based on confusion matrices showed in paired groups. The highest true positive rates (TPR) appeared between groups C vs D (93.5 % ± 2.7 %), groups C vs. DP (89.2 % ± 4.1 %), and groups D and P (90.4 % ± 3.2 %). However, P vs DP presented higher TPR for DP (84.1 % ±3.1 %) while D vs. DP the highest rate for DP was 81.7 % ± 4.3 %. Analyzing all groups together, the TPR decreased. CONCLUSION: The system used is portable and robust and can be widely used in clinical environments and hospitals as a new diagnostic technique. Studies in our groups are being conducted to solidify and expand data analysis methods with friendly language for healthcare professionals. It was possible to classify healthy patients in a range of 78-93 % of accuracy. Range over 80 % of accuracy between periodontitis and diabetes were observed. A general classification model with lower TPR instead of a pairwise classification would only have advantages in scenarios where no prior patient information is available regarding diabetes and periodontitis status.

Providing potential solutions by using FT-IR spectroscopy for biofluid analysis: Clinical impact of optical screening and diagnostic tests.

BACKGROUND: Currently, the potential of FT-IR spectroscopy for rapid diagnosis of many pathologies has been demonstrated by numerous research studies including those targeting COVID-19 detection. However, the number of clinicians aware of this potential and who are willing to use spectroscopy in their clinics and hospitals is still negligible. In addition, lack of awareness creates a huge gap between clinicians and researchers involved in clinical translation of current FT-IR technology hence hindering initiatives to bring basic and applied research together for the direct benefit of patients. METHODS: Knowledge and medical training on FT-IR on the side of clinicians should be one of the first steps to be able to integrate it into the list of complementary exams which may be requested by health professionals. Countless FT-IR applications could have a life-changing impact on patients' lives, especially screening and diagnostic tests involving biofluids such as blood, saliva and urine which are routinely non-invasively or minimally-invasively. RESULTS: Blood may be the most difficult to obtain by the invasive method of collection, but much can be evaluated in its components, and areas such as hematology, infectiology, oncology and endocrinology can be directly benefited. Urine with a relatively simple collection method can provide pertinent information from the entire urinary system, including the actual condition of the kidneys. Saliva collection can be simpler for the patient and can provide information on diseases affecting the mouth and digestive system and can be used to diagnose diseases such as oral cancer in its early-stages. An unavoidable second step is the active involvement of industries to design robust and portable instruments for specific purposes, as the medical community requires user-friendly instruments of advanced computational algorithms. A third step resides in the legal situation involving the global use of the technique as a new diagnostic modality. CONCLUSIONS: It is important to note that decentralized funds for variety of technologies hinders the training of clinical and medical professionals for the use of newly arising technologies and affect the engagement of these professionals with technology developers. As a result of decentralized funding, research efforts are spread out over a range of technologies which take a long time to get validated and translated to the clinic. Partnership over similar groups of technologies and efforts to test the same technologies while overcoming barriers posed to technology validation in different areas around the globe may benefit the clinical/medical, research and industry community globally.

Surface-Enhanced Raman Analysis of Uric Acid and Hypoxanthine Analysis in Fractionated Bodily Fluids.

In recent years, the disease burden of hyperuricemia has been increasing, especially in high-income countries and the economically developing world with a Western lifestyle. Abnormal levels of uric acid and hypoxanthine are associated with many diseases, and therefore, to demonstrate improved methods of uric acid and hypoxanthine detection, three different bodily fluids were analysed using surface-enhanced Raman spectroscopy (SERS) and high-performance liquid chromatography (HPLC). Gold nanostar suspensions were mixed with series dilutions of uric acid and hypoxanthine, 3 kDa centrifugally filtered human blood serum, urine and saliva. The results show that gold nanostars enable the quantitative detection of the concentration of uric acid and hypoxanthine in the range 5-50 µg/mL and 50-250 ng/mL, respectively. The peak areas of HPLC and maximum peak intensity of SERS have strongly correlated, notably with the peaks of uric acid and hypoxanthine at 1000 and 640 cm-1, respectively. The r2 is 0.975 and 0.959 for uric acid and hypoxanthine, respectively. Each of the three body fluids has a number of spectral features in common with uric acid and hypoxanthine. The large overlap of the spectral bands of the SERS of uric acid against three body fluids at spectra peaks were at 442, 712, 802, 1000, 1086, 1206, 1343, 1436 and 1560 cm-1. The features at 560, 640, 803, 1206, 1290 and 1620 cm-1 from hypoxanthine were common to serum, saliva and urine. There is no statistical difference between HPLC and SERS for determination of the concentration of uric acid and hypoxanthine (p > 0.05). For clinical applications, 3 kDa centrifugal filtration followed by SERS can be used for uric acid and hypoxanthine screening is, which can be used to reveal the subtle abnormalities enhancing the great potential of vibrational spectroscopy as an analytical tool. Our work supports the hypnosis that it is possible to obtain the specific concentration of uric acid and hypoxanthine by comparing the SER signals of serum, saliva and urine. In the future, the analysis of other biofluids can be employed to detect biomarkers for the diagnosis of systemic pathologies.

FTIR spectroscopy as a point of care diagnostic tool for diabetes and periodontitis: A saliva analysis approach.

Diabetes mellitus (DM) is a general term for heterogeneous metabolic disorders whose main characteristic is chronic hyperglycemia. Considering that conventional diagnostic methods are currently unable of early DM detection and the number of diabetic patients has been increasing worldwide, there is a clinical need for novel diagnostic approaches. Fourier-transform infrared (FT-IR) spectroscopy technique an alternative non-invasive diagnostic method for real-time evaluation of biofluids such as saliva. This study aims evaluate the feasibility of diagnosing diabetes and periodontitis through saliva samples based on their FT-IR spectra. Our first collection and spectral analysis of samples was a pilot study and comprised a total of 23 patients, 2 healthy, 9 with diabetes and 12 with diabetes and periodontitis. By using weighted KNN as classifier, we have obtained an area under the Receiver Operating Characteristic curve of 0.92 and 0.95 when considering the diabetes or diabetes + periodontitis groups as positive groups, respectively.

Label-Free Optical Spectroscopy for Early Detection of Oral Cancer.

Oral cancer is the 16th most common cancer worldwide. It commonly arises from painless white or red plaques within the oral cavity. Clinical outcome is highly related to the stage when diagnosed. However, early diagnosis is complex owing to the impracticality of biopsying every potentially premalignant intraoral lesion. Therefore, there is a need to develop a non-invasive cost-effective diagnostic technique to differentiate non-malignant and early-stage malignant lesions. Optical spectroscopy may provide an appropriate solution to facilitate early detection of these lesions. It has many advantages over traditional approaches including cost, speed, objectivity, sensitivity, painlessness, and ease-of use in clinical setting for real-time diagnosis. This review consists of a comprehensive overview of optical spectroscopy for oral cancer diagnosis, epidemiology, and recent improvements in this field for diagnostic purposes. It summarizes major developments in label-free optical spectroscopy, including Raman, fluorescence, and diffuse reflectance spectroscopy during recent years. Among the wide range of optical techniques available, we chose these three for this review because they have the ability to provide biochemical information and show great potential for real-time deep-tissue point-based in vivo analysis. This review also highlights the importance of saliva-based potential biomarkers for non-invasive early-stage diagnosis. It concludes with the discussion on the scope of development and future demands from a clinical point of view.

Evaluation of the Whitening Effectiveness of Violet Illumination Alone or Combined with Hydrogen Peroxide Gel.

Objective: To evaluate the effectiveness of the whitening and washing steps of a treatment using violet illumination (VI) alone or combined with hydrogen peroxide gel. In addition, we evaluated the color change after cleaning the tooth with and without mineral oil. Methods: First, 16 bovine teeth were extracted and stored in 5% thymol solution. Next, the teeth were collected and cleaned. Then, the teeth were stained with instant coffee solution for 24 h. The teeth were divided in four groups: control, VI without 35% hydrogen peroxide gel (VI), VI with 35% hydrogen peroxide gel (VI + gel), and VI without 35% hydrogen peroxide gel and cleaned with mineral oil before washing (VI + oil). Results: The whitening treatment VI + gel was able to completely restore the teeth whiteness and make the teeth 31.2% less yellow than prior coffee staining. The VI + oil treatment led to about 3.7 times the whiteness and yellowness changes observed in the VI treatment and restored 51% of the whiteness lost by staining. Conclusions: The VI + gel treatment can be recommended against coffee stains and should be further investigated for other types of tooth stains. In addition, cleaning the tooth surface with mineral oil could be an alternative to increase the performance of whitening treatments.

Evaluation of wavelength ranges and tissue depth probed by diffuse reflectance spectroscopy for colorectal cancer detection.

Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second most deadly. Recent research efforts have focused on developing non-invasive techniques for CRC detection. In this study, we evaluated the diagnostic capabilities of diffuse reflectance spectroscopy (DRS) for CRC detection by building 6 classification models based on support vector machines (SVMs). Our dataset consists of 2889 diffuse reflectance spectra collected from freshly excised ex vivo tissues of 47 patients over wavelengths ranging from 350 and 1919 nm with source-detector distances of 630-µm and 2500-µm to probe different depths. Quadratic SVMs were used and performance was evaluated using twofold cross-validation on 10 iterations of randomized training and test sets. We achieved (93.5 ± 2.4)% sensitivity, (94.0 ± 1.7)% specificity AUC by probing the superficial colorectal tissue and (96.1 ± 1.8)% sensitivity, (95.7 ± 0.6)% specificity AUC by sampling deeper tissue layers. To the best of our knowledge, this is the first DRS study to investigate the potential of probing deeper tissue layers using larger SDD probes for CRC detection in the luminal wall. The data analysis showed that using a broader spectrum and longer near-infrared wavelengths can improve the diagnostic accuracy of CRC as well as probing deeper tissue layers.

Diagnosis of Systemic Diseases Using Infrared Spectroscopy: Detection of Iron Overload in Plasma-Preliminary Study.

Despite the important role of iron in cellular homeostasis, iron overload (IO) is associated with systemic and tissue deposits which damage several organs. In order to reduce the impact caused by IO, invasive diagnosis exams (e.g., biopsies) and minimally invasive methods were developed including computed tomography and magnetic resonance imaging. However, current diagnostic methods are still time-consuming and expensive. A cost-effective solution is using Fourier-transform infrared spectroscopy (FTIR) for real-time and molecular-sensitive biofluid analysis during conventional laboratory exams. In this study, we performed the first evaluation of the accuracy of FTIR for IO diagnosis. The study was performed by collecting FTIR spectra of plasma samples of five rats intravenously injected with iron-dextran and five control rats. We developed a classification model based on principal component analysis and supervised methods including J48, random forest, multilayer perceptron, and radial basis function network. We achieved 100% accuracy for the classification of the IO status and provided a list of possible biomolecules related to the vibrational modes detected. In this preliminary study, we give a first step towards real-time diagnosis for acute IO or intoxication. Furthermore, we have expanded the literature knowledge regarding the pathophysiological changes induced by iron overload.

Rapid diagnosis of COVID-19 using FT-IR ATR spectroscopy and machine learning.

Early diagnosis of COVID-19 in suspected patients is essential for contagion control and damage reduction strategies. We investigated the applicability of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy associated with machine learning in oropharyngeal swab suspension fluid to predict COVID-19 positive samples. The study included samples of 243 patients from two Brazilian States. Samples were transported by using different viral transport mediums (liquid 1 or 2). Clinical COVID-19 diagnosis was performed by the RT-PCR. We built a classification model based on partial least squares (PLS) associated with cosine k-nearest neighbours (KNN). Our analysis led to 84% and 87% sensitivity, 66% and 64% specificity, and 76.9% and 78.4% accuracy for samples of liquids 1 and 2, respectively. Based on this proof-of-concept study, we believe this method could offer a simple, label-free, cost-effective solution for high-throughput screening of suspect patients for COVID-19 in health care centres and emergency departments.

Quantitative image of fluorescence of ceramic and resin-cement veneers.

The main of the study was quantify the effect of two ceramics with two underlying resin cements on apparent fluorescence levels. Buccal surfaces of two bovine incisors were ground flat producing one enamel and one dentin substrate. The veneers were fabricated (0.5 and 1.0 mm thickness) using two ceramics (IPSe.max Press and IPSe.max Zirpress, Ivoclar Vivadent). Veneers were cemented using either light-cured (Variolink II, Ivoclar Vivadent) or self-adhesive dual (Rely X U200, 3M ESPE) cement. The layered Control group materials had no cement application. Semi-quantitative fluorescence image analysis (Matlabs software, Matworks) involved processing the images as captured under each daylight (DL, Gretagmacbeth) and ultraviolet illuminants (UVA, Sylvania) within a neutral-gray lightbox (Macbeth Spectral Light). Statistical analysis of the quantitative fluorescence values was performed using two-way ANOVA and Tukey's test (p < 0.05). The e.max Zirpress on the dentin substrate produced greater fluorescence (p < 0.05) when subjected to UV illumination and more fluorescence (p < 0.05) than e.max Press in both cement groups. Light-cured cement produced higher (p < 0.05) fluorescence than the dual-cement with e.max Press on enamel under UV illumination. The fluorescence for e.max Press on the dentin substrate was greater (p < 0.05) than for e.max Zirpress using dual self-adhesive cement subjected to daylight illumination. Thus, it is possible to conclude that the combination of ceramic and cement produce definite, significant effects on the apparent fluorescence, vital quality for restorative dentistry.

Erratum: Time-resolved fluorescence spectroscopy for clinical diagnosis of actinic cheilitis: erratum.

[This corrects the article on p. 4210 in vol. 7, PMID: 27867726.].

Erratum: Raman spectral post-processing for oral tissue discrimination - a step for an automatized diagnostic system: erratum.

[This corrects the article on p. 5218 in vol. 8, PMID: 29188115.].

Raman spectral post-processing for oral tissue discrimination - a step for an automatized diagnostic system.

Most oral injuries are diagnosed by histopathological analysis of a biopsy, which is an invasive procedure and does not give immediate results. On the other hand, Raman spectroscopy is a real time and minimally invasive analytical tool with potential for the diagnosis of diseases. The potential for diagnostics can be improved by data post-processing. Hence, this study aims to evaluate the performance of preprocessing steps and multivariate analysis methods for the classification of normal tissues and pathological oral lesion spectra. A total of 80 spectra acquired from normal and abnormal tissues using optical fiber Raman-based spectroscopy (OFRS) were subjected to PCA preprocessing in the z-scored data set, and the KNN (K-nearest neighbors), J48 (unpruned C4.5 decision tree), RBF (radial basis function), RF (random forest), and MLP (multilayer perceptron) classifiers at WEKA software (Waikato environment for knowledge analysis), after area normalization or maximum intensity normalization. Our results suggest the best classification was achieved by using maximum intensity normalization followed by MLP. Based on these results, software for automated analysis can be generated and validated using larger data sets. This would aid quick comprehension of spectroscopic data and easy diagnosis by medical practitioners in clinical settings.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin.

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis.

Time-resolved fluorescence spectroscopy for clinical diagnosis of actinic cheilitis.

Actinic cheilitis is a potentially malignant disorder of the lips. Its first cause is believed to be UV sun radiation. The lesion is highly heterogeneous, making the choice of area to be biopsied difficult. This study exploits the capabilities of time-resolved fluorescence spectroscopy for the identification of the most representative area to be biopsied. A preliminary study was performed on fourteen patients. A classification algorithm was used on data acquired on nine different biopsies. The algorithm discriminated between absent, mild, and moderate dysplasia with a sensitivity of 92.9%, 90.0%, and 80.0%, respectively. The false positive rate for healthy tissue (specificity) was 88.8%.