Vibrational spectroscopic detection of radiation-induced structural changes in Chironomus hemoglobin.

Purpose: Chironomus hemoglobin is known to exhibit higher gamma radiation resistance compared to human hemoglobin. In the present study, we have introduced a sensitive method to analyze radiation-induced alterations in Chironomus hemoglobin using Vibrational spectroscopy and further highlighting its potential for monitoring radiotoxicity in aquatic environments. Materials and methods: Vibrational spectroscopic methods such as Raman and FT-IR spectroscopy were used to capture the distinctive chemical signature of Chironomus hemoglobin (ChHb) under both in vitro and in vivo conditions. Any radiation dose-dependent shifts could be analyzed Human hemoglobin (HuHb) as standard reference. Results: Distinctive Raman peak detected at 930 cm-1 in (ChHb) was attributed to C-N stretching in the heterocyclic ring surrounding the iron atom, preventing heme degradation even after exposure to 2400 Gy dose. In contrast, for (HuHb), the transition from deoxy-hemoglobin to met-hemoglobin at 1210 cm-1 indicated a disruption in oxygen binding after exposure to 1200 Gy dose. Furthermore, while ChHb exhibited a consistent peak at 1652 cm-1 in FT-IR analysis, HuHb on the other hand, suffered damage after gamma irradiation. Conclusion: The findings suggest that vibrational spectroscopic methods hold significant potential as a sensitive tool for detecting radiation-induced molecular alterations and damages. Chironomus hemoglobin, with its robust interaction of the pyrrole ring with Fe, serves as a reliable bioindicator molecule to detect radiation damage using vibrational spectroscopic method.

Serum Raman spectroscopy: Evaluation of tumour load variations in experimental carcinogenesis.

Several serum Raman spectroscopy (RS) studies have demonstrated its potential as an oral cancer screening tool. This study investigates influence of low tumour load (LTL) and high tumour load (HTL) on serum RS using hamster buccal pouch model of experimental oral carcinogenesis. Sera of untreated control, LTL, and HTL groups at week intervals during malignant transformation were employed. Serum Raman spectra were subjected to multivariate analyses-principal component analysis, principal component-based linear discriminant analysis (for stratification of study groups), and multivariate curve resolution-alternating least squares (MCR-ALS) (to comprehend biomolecular differences). Multivariate analysis revealed misclassifications between LTL and HTL at all week intervals. MCR-ALS components showed statistically significant abundances between control versus LTL and control versus HTL, but could not discern LTL and HTL. MCR-ALS components exhibited spectral mixtures of proteins, lipids, heme and nucleic acids. Thus, these findings support use of serum RS as a screening tool as varying tumour load is not a confounding factor influencing the technique.

Serum Raman spectroscopy: Prognostic applications in oral cancers.

BACKGROUND: Loco-regional recurrences attributable to field cancerization and minimal residual cancer, remain prime causes of mortality in oral cancer (OC) subjects. The current study evaluates potential of serum Raman spectroscopy (SRS) to identify recurrence-prone OC subjects. METHODS: Raman spectra of serum from eight healthy subjects (H) and 57 OC subjects (with-recurrence [R], without-recurrence [NR], and with suspicious-lesions [S]), before (BS) and after (AS) surgical excision of tumor were recorded. OC subjects were followed-up for 7-years. RESULTS: DNA and protein alterations were observed in AS sera of all groups. 4-, 3-, and 2-model multivariate analyses were used to stratify BS and AS groups. H spectra were 100% distinguishable from all other groups. AS, R and NR were distinguished with high accuracy (84%) in all models. No stratification (~50%) was observed BS. CONCLUSION: SRS shows potential to identify recurrence prone subjects, post-surgery, using serum collected as early as 1 week after surgery.

SARS-CoV-2 severity classification from plasma sample using confocal Raman spectroscopy.

The world is on the brink of facing coronavirus's (COVID-19) fourth wave as the mutant forms of viruses are escaping neutralizing antibodies in spite of being vaccinated. As we have already witnessed that it has encumbered our health system, with hospitals swamped with infected patients observed during the viral outbreak. Rapid triage of patients infected with SARS-CoV-2 is required during hospitalization to prioritize and provide the best point of care. Traditional diagnostics techniques such as RT-PCR and clinical parameters such as symptoms, comorbidities, sex and age are not enough to identify the severity of patients. Here, we investigated the potential of confocal Raman microspectroscopy as a powerful tool to generate an expeditious blood-based test for the classification of COVID-19 disease severity using 65 patients plasma samples from cohorts infected with SARS-CoV-2. We designed an easy manageable blood test where we used a small volume (8 µl) of inactivated whole plasma samples from infected patients without any extra solvent usage in plasma processing. Raman spectra of plasma samples were acquired and multivariate exploratory analysis PC-LDA (principal component based linear discriminant analysis) was used to build a model, which segregated the severe from the non-severe COVID-19 group with a sensitivity of 83.87%, specificity of 70.60% and classification efficiency of 76.92%. Among the bands expressed in both the cohorts, the study led to the identification of Raman fingerprint regions corresponding to lipids (1661, 1742), proteins amide I and amide III (1555, 1247), proteins (Phe) (1006, 1034), and nucleic acids (760) to be differentially expressed in severe COVID-19 patient's samples. In summary, the current study exhibits the potential of confocal Raman to generate simple, rapid, and less expensive blood tests to triage the severity of patients infected with SARS-CoV-2.

Photothermal therapy (PTT) is an effective treatment measure against solid tumors which fails to respond conventional chemo/radiation therapies in clinic.

Photothermal therapy (PTT) has emerged as a fast, precisive, and cost-effective anticancer therapy protocol. Here we applied our previously designed nanomaterial (Tocophotoxil) for prospective PTT application to manage radiation- and chemo-resistant cancers in a preclinical model. A PTT dose vs. efficacy relationship was established for radioresistant breast (ZR-75-1 50Gy, 4T1 20Gy) and chemo-resistant ovarian (A2780LR) cancer cells and tumors in mice models. Compared to the sensitive cases, resistant cells treated with PTT for a shorter duration show higher endurance. However, preclinical tumor xenografts treated with optimal PTT dose show 2-3 fold higher longevity (P ≤ 0.05) of treated mice monitored by non-invasive imaging methods. Elevated ERK and AKT activation in radioresistant or only AKT activation in chemo-resistant cells were contributory to higher cell survival in sub-optimal PTT dose. A comprehensive single-cell Raman map of PTT treated ZR-75-1 cell reveals broad-spectrum macromolecular deformities, including protein damage features. Marked induction of pJNK, unfolded protein response (UPR) pathway, increased reactive oxygen species (ROS), and lipid peroxidation in PTT-treated cells disrupted the intracellular homeostasis. Analyzing cellular ultrastructure, the coexistence of swollen endoplasmic reticulum, and autophagic bodies after PTT indicate possible coordination between UPR and autophagy pathways. Therefore, this comprehensive study provides new evidence on the potential impact of PTT as a standalone therapy for ablation of failed conventional therapy-resistant cancers in vivo, the success of which is intricately linked to the PTT dose optimization. The study, for the first time, also illustrates that under PTT treatment, concerted action of novel molecular switches such as JNK activation and UPR activation plays a vital role in triggering autophagy and cancer cell death.

Improving Vibrational Spectroscopy Prospects in Frontline Clinical Diagnosis: Fourier Transform Infrared on Buccal Mucosa Cancer.

We report a novel method with higher than 90% accuracy in diagnosing buccal mucosa cancer. We use Fourier transform infrared spectroscopic analysis of human serum by suppressing confounding high molecular weight signals, thus relatively enhancing the biomarkers' signals. A narrower range molecular weight window of the serum was also investigated that yielded even higher accuracy on diagnosis. The most accurate results were produced in the serum's 10-30 kDa molecular weight region to distinguish between the two hardest to discern classes, i.e., premalignant and cancer patients. This work promises an avenue for earlier diagnosis with high accuracy as well as greater insight into the molecular origins of these signals by identifying a key molecular weight region to focus on.

Unraveling the Secrets of Colistin Resistance with Label-Free Raman Spectroscopy.

The rise in number of infections from multidrug-resistant (MDR) Gram-negative microbes has led to an increase in the use of a variety of 'polymyxins' such as colistin. Even though colistin is known to cause minor nephro- and neuro-toxicity, it is still considered as last resort antibiotic for treating MDR infections. In this study, we have applied Raman spectroscopy to understand the differences among colistin sensitive and resistant bacterial strains at community level. We have successfully generated colistin resistant clones and verified the presence of resistance-causing MCR-1 plasmid. A unique spectral profile associated with specific drug concentration has been obtained. Successful delineation between resistant and sensitive cells has also been achieved via principal component analysis. Overall findings support the prospective utility of Raman spectroscopy in identifying anti-microbial resistance.

Metabolomics Profiling of Pituitary Adenomas by Raman Spectroscopy, Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy, and Mass Spectrometry of Serum Samples.

To date, no studies are available in which pituitary adenomas (PAs) have been studied using techniques like confocal Raman spectroscopy, attenuated total reflection-Fourier transform infrared (FT-IR), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the same serum samples. To understand the metabolomics fingerprint, Raman spectra of 16 acromegaly, 19 Cushing's, and 33 nonfunctional PA (NFPA) and ATR-FTIR spectral acquisition of 16 acromegaly, 18 Cushing's, and 22 NFPA patient's serum samples were acquired. Next, Principal component-based linear discriminant analysis (PC-LDA) models were developed, Raman spectral analysis classified acromegaly with an accuracy of 79.17%, sensitivity of 75%, and specificity of 81.25%, Cushing's with an accuracy of 66.67%, sensitivity of 100%, and specificity of 52.63%, and NFPA with an accuracy of 73.17%, sensitivity of 75%, and specificity of 72.73%. ATR-FTIR spectral analysis classified acromegaly with an accuracy of 95.83%, sensitivity of 100%, and specificity of 93.75%, Cushing's with an accuracy of 65.38%, sensitivity of 87.5%, and specificity of 55.56%, and NFPA with an accuracy of 70%, sensitivity of 87.5%, and specificity of 43.75%. In either of the cases, healthy individual cohorts were clearly segregated from the disease cohort, which identified differential regulated regions of nucleic acids, lipids, amides, phosphates, and polysaccharide/C-C residue α helix regions. Furthermore, LC-MS/MS-based analysis of sera samples resulted in the identification of various sphingosine, lipids, acylcarnitines, amino acids, ethanolamine, choline, and their derivatives that differentially regulated in each tumor cohort. We believe cues obtained from the study may be used to generate the metabolite-based test to diagnose PAs from serum in addition to conventional techniques and also to understand disease biology for better disease management, point of care, and improving quality of life in PA patients.

Growth Kinetics Monitoring of Gram-Negative Pathogenic Microbes Using Raman Spectroscopy.

Optical density based measurements are routinely performed to monitor the growth of microbes. These measurements solely depend upon the number of cells and do not provide any information about the changes in the biochemical milieu or biological status. An objective information about these parameters is essential for evaluation of novel therapies and for maximizing the metabolite production. In the present study, we have applied Raman spectroscopy to monitor growth kinetics of three different pathogenic Gram-negative microbes Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Spectral measurements were performed under 532 nm excitation with 5 seconds of exposure time. Spectral features suggest temporal changes in the "peptide" and "nucleic acid" content of cells under different growth stages. Using principal component analysis (PCA), successful discrimination between growth phases was also achieved. Overall, the findings are supportive of the prospective adoption of Raman based approaches for monitoring microbial growth.

ß-Lactoglobulin-gold nanoparticles interface and its interaction with some anticancer drugs - an approach for targeted drug delivery.

The protein-nanoparticle interface plays a crucial role in drug binding and stability, in turn enhancing efficacy in targeted drug delivery. In the present study, whey protein ß-lactoglobulin (BLG) is conjugated with gold nanoparticles (AuNP) and its interaction with curcumin (CUR) and gemcitabine (GEM) has been explored. Further, AuNP-BLG conjugate interactions with anticancer drugs were characterized using dynamic light scattering (DLS), zeta potential, UV-visible, Raman spectroscopy, fluorescence, circular dichroism along with molecular dynamics simulation. The cytotoxicity studies were performed using breast cancer cell lines (MCF-7). ∼8 µM of BLG resides on AuNP (∼29 nm) surface revealed by DLS. Raman scattering of AuNP-BLG conjugate showed orientation of the central calyx of BLG towards solvent. BLG fluorescence confirmed the interaction between AuNP-BLG conjugate with drugs and indicated strong binding and affinity (for CUR KD = 3.71 x 108 M -1, n = 1.83, and for GEM KD = 3.78 x 103 M -1, n = 0.94), enhanced in the presence of AuNP. CD and Raman analysis exhibited selective hydrophilic and hydrophobic conformations induced by drug binding. Computational studies on BLG-drug complexes revealed that the residues Pro38, Leu39 and Met107 are largely associated with CUR binding, while GEM interaction is via hydrophilic contacts which significantly matches with spectroscopic investigation. IC50 values were calculated for all components of this loading system on MCF-7. The possible mechanisms of interaction between AuNP-BLG with anticancer drugs has been explored at the molecular level. We believe that these conjugates could be considered in the targeted drug delivery studies for cancer research.Communicated by Ramaswamy H. Sarma.

DNA Fingerprint Analysis of Raman Spectra Captures Global Genomic Alterations in Imatinib-Resistant Chronic Myeloid Leukemia: A Potential Single Assay for Screening Imatinib Resistance.

Monitoring the development of resistance to the tyrosine kinase inhibitor (TKI) imatinib in chronic myeloid leukemia (CML) patients in the initial chronic phase (CP) is crucial for limiting the progression of unresponsive patients to terminal phase of blast crisis (BC). This study for the first time demonstrates the potential of Raman spectroscopy to sense the resistant phenotype. Currently recommended resistance screening strategy include detection of BCR-ABL1 transcripts, kinase domain mutations, complex chromosomal abnormalities and BCR-ABL1 gene amplification. The techniques used for these tests are expensive, technologically demanding and have limited availability in resource-poor countries. In India, this could be a reason for more patients reporting to clinics with advanced disease. A single method which can identify resistant cells irrespective of the underlying mechanism would be a practical screening strategy. During our analysis of imatinib-sensitive and -resistant K562 cells, by array comparative genomic hybridization (aCGH), copy number variations specific to resistant cells were detected. aCGH is technologically demanding, expensive and therefore not suitable to serve as a single economic test. We therefore explored whether DNA finger-print analysis of Raman hyperspectral data could capture these alterations in the genome, and demonstrated that it could indeed segregate imatinib-sensitive and -resistant cells. Raman spectroscopy, due to availability of portable instruments, ease of spectrum acquisition and possibility of centralized analysis of transmitted data, qualifies as a preliminary screening tool in resource-poor countries for imatinib resistance in CML. This study provides a proof of principle for a single assay for monitoring resistance to imatinib, available for scrutiny in clinics.

Raman micro-spectroscopic map estimating in vivo precision of tumor ablative effect achieved by photothermal therapy procedure.

Photothermal-therapy (PTT) inculcates near-infrared laser guided local heating effect, where high degree of precision is expected, but not well proven to-date. An ex vivo tissue biochemical map with molecular/biochemical response showing the coverage area out of an optimized PTT procedure can reveal precision information. In this work, Raman-microscopic mapping and linear discriminant analysis of spectra of PTT treated and surrounding tissue areas ex vivo are done, revealing three distinct spectral clusters/zones, with minimal overlap between the core treated and adjacent untreated zone. The core treated zone showed intense nucleic-acid, cytochrome/mitochondria and protein damage, an adjacent zone showed lesser degree of damages and far zone showed minimal/no damage. Immunohistochemistry for γH2AX (DNA damage marker protein) in PTT exposed tissue also revealed similar results. Altogether, this study reveals the utility of Raman-microspectroscopy for fine-tuning safety parameters and precision that can be achieved from PTT mediated tumor ablation in preclinical/clinical application.

Risk prediction by Raman spectroscopy for disease-free survival in oral cancers.

In the present study, the potential of Raman spectroscopy (RS) in predicting disease-free survival (DFS) in oral cancer patients has been explored. Raman spectra were obtained from the tumor and contralateral regions of 94 oral squamous cell carcinoma patients. These patients were managed surgically and recommended for adjuvant therapy. The Cox proportional survival analysis was carried out to identify the spectral regions that can be correlated to DFS. The survival analysis was performed with 95% confidence intervals, hazard ratio, and p-values in the 1200-1800 cm-1 spectral region. Out of a total of 182 spectral points, 76 were found to be correlating with DFS, suggesting their utility to predict the patient outcome. The cut-off points of each correlating RS-point values were defined and tested towards predicting the DFS. The performance of predicting the power of spectral points was validated through Brier value, and it was found to be closer to the actual progression. The 76 spectral points identified from the tumors have the potential to accurately predict DFS in oral squamous cell carcinoma through a relatively simplistic prediction model in the absence of confounding factors.

Distinct stratification of normal liver, hepatocellular carcinoma (HCC), and anticancer nanomedicine-treated- tumor tissues by Raman fingerprinting for HCC therapeutic monitoring.

Hepatocellular carcinomas (HCCs) are highly vascularized neoplasms with poor prognosis. Nanomedicine possesses great potential to deliver therapeutics and diagnostics. The new aspect of this study is that we have monitored, for the first time, the Raman responses to microtubule targeted vascular disrupting agents (MTVDA), MTVDA encapsulated non-targeted, and targeted cetuximab polymeric nanocomplexes delivery of combinatorial therapeutics in HCC tumor tissues of mice. Biochemical differences majorly demarcated apoptotic lipid bodies, and characteristic amide-I features. HCC tumor and healthy liver tissues could be stratified. Raman spectroscopy served as an excellent, rapid, sensitive and cost-effective approach for anticancer nanomedicine distinct stratification of MTVDA encapsulated targeted cetuximab polymeric nanocomplex combinatorials, a significant potential for HCC therapeutic monitoring.

Salivary Raman Spectroscopy: Standardization of Sampling Protocols and Stratification of Healthy and Oral Cancer Subjects.

Minimally invasive cancer detection using bio-fluids has been actively pursued due to practical limitations, though there are better suited noninvasive and online in vivo methods. Saliva is one such clinically informative bio-fluid that offers the advantages of easy and multiple sample collection. Despite its potential in cancer diagnostics, saliva analysis is challenging due to its heterogeneous composition. Recently, there has been an upsurge in saliva exploration using optical techniques. Forms of saliva such as precipitate and supernatant have been monitored, but this sampling method needs to be standardized due to the obvious loss of analytes in processing. In that context, present work details the comparison of four different saliva sampling methodologies, i.e., air-dried, lyophilized, pellet, and supernatant using Raman spectroscopy collected from 10 healthy samples. Composition-driven spectral features of all forms were compared and classified using principal component analysis and linear discriminant analysis. Analysis was carried out on all four groups in the first step. In the second step, groups of pellet and supernatant , and air-dried and lyophilized were analyzed. Findings suggest that pellet and supernatant exhibit discrete spectroscopic features and demonstrate high classification efficiency, which is indicative of their distinctive biochemical composition. On the other hand, air-dried and lyophilized forms showed overlapping spectral features and low classification, suggesting these forms retain majority spectroscopic features of whole saliva and are less prone to sampling losses. Thus, this study indicates air-dried and lyophilized forms may be more appropriate for saliva sampling using Raman spectroscopy providing the comprehensive information required for cancer diagnosis. Furthermore, the method was also tested for the classification of oral cancer and healthy subjects (n = 27) which yielded 90% stratification. The findings of the study indicate the utility of minimally invasive salivary Raman-based diagnostics in oral cancers.

Raman spectroscopy-based detection of RNA viruses in saliva: A preliminary report.

Several non-invasive Raman spectroscopy-based assays have been reported for rapid and sensitive detection of pathogens. We developed a novel statistical model for the detection of RNA viruses in saliva, based on an unbiased selection of a set of 65 Raman spectral features that mostly attribute to the RNA moieties, with a prediction accuracy of 91.6% (92.5% sensitivity and 88.8% specificity). Furthermore, to minimize variability and automate the downstream analysis of the Raman spectra, we developed a GUI-based analytical tool "RNA Virus Detector (RVD)." This conceptual framework to detect RNA viruses in saliva could form the basis for field application of Raman Spectroscopy in managing viral outbreaks, such as the ongoing COVID-19 pandemic. (http://www.actrec.gov.in/pi-webpages/AmitDutt/RVD/RVD.html).

Raman Spectroscopy: An Exploratory Study to Identify Post-Radiation Cell Survival.

Resistance to radiotherapy has been an impediment in the treatment of cancer, and the inability to detect it at an early stage further exacerbates the prognosis. We have assessed the feasibility of Raman spectroscopy as a rapid assay for predicting radiosensitivity of cancer cells in comparison to the conventional biological assays. Cell lines derived from breast adenocarcinoma (MCF7), gingivobuccal squamous cell carcinoma (ITOC-03), and human embryonic kidney (HEK293) were subjected to varying doses of ionizing radiation. Cell viability of irradiated cells was assessed at different time points using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Raman spectroscopy, and colony-forming capability was evaluated by clonogenic assay. Radiosensitivity observed using MTT assay was limited by the finding of similar cell viability in all the three cell lines 24 h post-irradiation. However, cell survival assessed using clonogenic assay and principal component linear discriminant analysis (PC-LDA) classification of Raman spectra showed correlating patterns. Irradiated cells showed loss of nucleic acid features and enhancement of 750 cm-1 peak probably attributing to resonance Raman band of cytochromes in all three cell lines. PC-LDA analysis affirmed MCF7 to be a radioresistant cell line as compared to ITOC-03 and HEK293 to be the most radiosensitive cell line. Raman spectroscopy is shown to be a rapid and alternative assay for identification of radiosensitivity as compared to the gold standard clonogenic assay.

Quality assessment of cryopreserved biospecimens reveals presence of intact biomolecules.

Recapitulation of tumor features in isolated biomolecules is preeminently dependent on obtaining reliable quality biospecimen. Moreover, quality assessment of biobanked specimens at regular intervals is an essential intervention for carrying out effective translational and clinical research. In the current study, genomic DNA was extracted from 140 fresh frozen tissues of oral, breast and colorectal specimens cryopreserved over a period of 3 to 8 months (short term) and 3 to 4 years (long term). Quantification of genomic DNA by absorption and fluorescence spectroscopy confirmed high concentration while qualitative analysis by gel electrophoresis showed intact bands for 94% and 87% of short- and long-term cohorts, respectively. PC-LDA based classification of Raman spectra showed overlapping groups of both cohorts suggesting the quality of DNA being preserved irrespective of storage period. To the best of our knowledge this is the first Indian biobank study reporting quality analysis of biospecimens cryopreserved at different time periods.

Unique spectral markers discern recurrent Glioblastoma cells from heterogeneous parent population.

An inability to discern resistant cells from bulk tumour cell population contributes to poor prognosis in Glioblastoma. Here, we compared parent and recurrent cells generated from patient derived primary cultures and cell lines to identify their unique molecular hallmarks. Although morphologically similar, parent and recurrent cells from different samples showed variable biological properties like proliferation and radiation resistance. However, total RNA-sequencing revealed transcriptional landscape unique to parent and recurrent populations. These data suggest that global molecular differences but not individual biological phenotype could differentiate parent and recurrent cells. We demonstrate that Raman Spectroscopy a label-free, non-invasive technique, yields global information about biochemical milieu of recurrent and parent cells thus, classifying them into distinct clusters based on Principal-Component-Analysis and Principal-Component-Linear-Discriminant-Analysis. Additionally, higher lipid related spectral peaks were observed in recurrent population. Importantly, Raman spectroscopic analysis could further classify an independent set of naïve primary glioblastoma tumour tissues into non-responder and responder groups. Interestingly, spectral features from the non-responder patient samples show a considerable overlap with the in-vitro generated recurrent cells suggesting their similar biological behaviour. This feasibility study necessitates analysis of a larger cohort of naïve primary glioblastoma samples to fully envisage clinical utility of Raman spectroscopy in predicting therapeutic response.

Raman mapping of oral buccal mucosa: a spectral histopathology approach.

Oral cancer is one of the most common cancers worldwide. One-fifth of the world's oral cancer subjects are from India and other South Asian countries. The present Raman mapping study was carried out to understand biochemical variations in normal and malignant oral buccal mucosa. Data were acquired using WITec alpha 300R instrument from 10 normal and 10 tumors unstained tissue sections. Raman maps of normal sections could resolve the layers of epithelium, i.e. basal, intermediate, and superficial. Inflammatory, tumor, and stromal regions are distinctly depicted on Raman maps of tumor sections. Mean and difference spectra of basal and inflammatory cells suggest abundance of DNA and carotenoids features. Strong cytochrome bands are observed in intermediate layers of normal and stromal regions of tumor. Epithelium and stromal regions of normal cells are classified by principal component analysis. Classification among cellular components of normal and tumor sections is also observed. Thus, the findings of the study further support the applicability of Raman mapping for providing molecular level insights in normal and malignant conditions.