Autofluorescence Spectroscopy Research Articles

Autofluorescence Signatures of Seven Pathogens: Preliminary in Vitro Investigations of a Potential Diagnostic for Acanthamoeba Keratitis.

Acanthamoeba keratitis can cause devastating damage to the human cornea and is often difficult to diagnose by routine clinical methods. In this preliminary study, we investigated whether Acanthamoeba may be distinguished from other common corneal pathogens through its autofluorescence response. Although only a small number of pathogens were studied, the identification of a unique Acanthamoeba signature would indicate that autofluorescence spectroscopy as a diagnostic method merits further investigation. Samples of 7 common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Elizabethkingia miricola, Achromobacter ruhlandii, Candida albicans, and Acanthamoeba castellanii) in solution were excited with ultraviolet light at a number of successive, narrow wavebands between 260 and 400 nm, and their fluorescence response recorded. Principal Component Analysis was used to allow better visualization of the differences in response to UV light for different species. Acanthamoeba was found to possess a characteristic autofluorescence response and was easily distinguished from E. coli, S. aureus, P. aeruginosa, E. miricola, A. ruhlandii, and C. albicans over a wide range of excitation wavelengths. We also found a clear discrimination between E. coli, C. albicans, and P. aeruginosa at an excitation wavelength of 274 nm, whereas E. miricola, S. aureus, and A. ruhlandii could be separated using an excitation wavelength of 308 nm. Our results, although preliminary, indicate that autofluorescence spectroscopy shows promise as a diagnostic technique for keratitis. We intend to expand the set of pathogens studied before assessing the feasibility of the technique in vivo by introducing cultures onto pig corneas.

Near-infrared autofluorescence spectroscopy of in vivo soft tissue sarcomas

Soft tissue sarcomas (STS) are a rare and heterogeneous group of malignant tumors that are often treated via surgical resection. Inadequate resection can lead to local recurrence and decreased survival rates. In this study, we investigate the hypothesis that near-infrared (NIR) autofluorescence can be utilized for tumor margin analysis by differentiating STS from the surrounding normal tissue. Intraoperative in vivo measurements were acquired from 30 patients undergoing STS resection and were characterized to differentiate between normal tissue and STS. Overall, normal muscle and fat were observed to have the highest and lowest autofluorescence intensities, respectively, with STS falling in between. With the exclusion of well-differentiated liposarcomas, the algorithm's accuracy for classifying muscle, fat, and STS was 93%, 92%, and 88%, respectively. These findings suggest that NIR autofluorescence spectroscopy has potential as a rapid and nondestructive surgical guidance tool that can inform surgeons of suspicious margins in need of immediate re-excision.

Establishing the clinical utility of autofluorescence spectroscopy for parathyroid detection

The inability of surgeons to identify parathyroid glands accurately during cervical endocrine surgery hinders patients from achieving postoperative normocalcemia. An intrinsic, near-infrared fluorescence method was developed for real-time parathyroid identification with high accuracy. This study assesses the clinical utility of this approach. Autofluorescence measurements were obtained from 137 patients (264 parathyroid glands) undergoing parathyroidectomy and/or thyroidectomy. Measurements were correlated to disease state, calcium levels, parathyroid hormone, vitamin D levels, age, sex, ethnicity, and body mass index. Statistical analysis identified which factors affect parathyroid detection. High parathyroid fluorescence was detected consistently and showed wide variability across patients. Near-infrared fluorescence was used to identify 256 of 264 (97%) of glands correctly. The technique showed high accuracy over a wide variety of disease states, although patients with secondary hyperparathyroidism demonstrated confounding results. Analysis revealed body mass index (P < .01), disease state (P < .01), vitamin D (P < .05), and calcium levels (P < .05) account greatly for variability in signal intensity. Age, sex, parathyroid hormone, and ethnicity had no effect. This intrinsic fluorescence-based intraoperative technique can detect nearly all parathyroid glands accurately in real time. Its discrimination capacity is largely unlimited by patient variables, but several factors affect signal intensity. These results demonstrate potential clinical utility of optical guidance for parathyroid detection.

165 Label-free autofluorescence lifetime to assess changes in myocardial fibrosis and metabolismin vivoin a doxorubicin cardiomyopathy heart failure model

We are developing an autofluorescence (AFL) lifetime-based technique to characterise the signatures associated with histological, metabolic and functional changes in myocardial disease. AFL spectroscopy exploits the properties of a number of endogenous molecules and offers the potential to avoid exogenous label use that could potentially alter the physiological environment. AFL measurements of molecules such as NADH and flavins may report energetic state changes, whilst signatures from matrix such as collagen inform of structural alterations. We investigated the application of a fibre-optic based single-point instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to a doxorubicin cardiomyopathy heart failure model (DOX-HF) in vivo . Sprague-Dawley male rats received 1.25 mg/kg doxorubicin or vehicle (0.9% NaCl) via tail vein injection weekly for 8 weeks. AFL signals were measured in vivo in week 11, once heart failure phenotype was well established. DOX-HF(n = 8) LVEF 49.6% vs. 80.4% in controls (n = 4)(**p = 0.006). At 11 weeks, significant differences in AFL signals between DOX-HF(n = 6) and control (n = 3) in LV, RV and LV posterior wall were observed (*p Our instrument has sensitivity to characterise in vivo changes in DOX-HF model without use of exogenous compounds. Incorporation into coronary catheters or pacing leads could offer additional diagnostic information and monitoring. Identification of earlier changes could permit development of a screening strategy to identify cancer patients developing cardiomyopathy to permit judicious adjustment of chemotherapy.

Freshness estimation of intact frozen fish using fluorescence spectroscopy and chemometrics of excitation–emission matrix

The current study attempted to provide a convenient, non-invasive and time-saving method to estimate the freshness of intact horse mackerel (Trachurus japonicus) fish in a frozen state using autofluorescence spectroscopy in tandem with multivariate analysis of fluorescence excitation–emission matrices (EEM). The extracted fluorescence data from different freshness conditions were pretreated, masked and reorganized to resolve fish fluorescence spectra from overlapping signals and scattering profiles for detecting and characterizing freshness changes. The real freshness values of the examined fish samples were then traditionally determined by the hard chemical analysis using the high performance liquid chromatography (HPLC) method and expressed as K-values. The fluorescence EEM data and the real freshness values were modeled using partial least square (PLS) regression and a novel algorithm was proposed to identify the ideal combinations of excitation and emission wavelengths being used as perfect predictors. The results revealed that freshness of frozen fish could be accurately predicted with R2 of 0.89 and root mean square error estimated by cross validation (RMSECV) of 9.66%. This work substantially demonstrated that the autofluorescence spectroscopy associated with the proposed technical approaches has a high potential in non-destructive sensing of fish freshness in the frozen state.

Detection and quantification of dental plaque based on laser-induced autofluorescence intensity ratio values.

The aim of this study was to evaluate the applicability of laser-induced autofluorescence (LIAF) spectroscopy to detect and quantify dental plaque. LIAF spectra were recorded in situ from dental plaque (0–3 grades of plaque index) in 300 patients with 404 nm diode laser excitation. The fluorescence intensity ratio of the emission peaks was calculated from the LIAF spectral data following which their scatter plots were drawn and the area under the receiver operating characteristics were calculated. The LIAF spectrum of clinically invisible grade-1 plaque showed a prominent emission peak at 510 nm with a satellite peak around 630 nm in contrast to grade 0 that has a single peak around 500 nm. The fluorescence intensity ratio (F510/F630) has a decreasing trend with increase in plaque grade and the ratio values show statistically significant differences (p<0.01) between different grades. An overall sensitivity and specificity of 100% each was achieved for discrimination between grade-0 and grade-1 plaque. The clinical significance of this study is that the diagnostic algorithm developed based on fluorescence spectral intensity ratio (F510/F630) would be useful to precisely identify minute amounts of plaque without the need for disclosing solutions and to convince patients of the need for proper oral hygiene and homecare practices.

Laser-induced autofluorescence spectroscopy: Can it be of importance in detection of bladder lesions?

Non-muscle invasive bladder cancer can be missed during white light endoscopy in up to 50% of cases. We aimed to test whether or not we could find a difference between benign and cancerous tissue wavelengths using laser induced autofluorescence spectroscopy can increase cancer detection. We analysed 67 tissue samples using spectral analysis. The WavSTAT (Spectra Science) optical biopsy device was used to record fluorescence spectra from biopsied tissue enabling calculation of an AUC for each spectrum, a measure of the mean spectral wavelength (λ¯ (nm)) and a dimensionless fluorescence ratio. Mann-Whitney test was used to compare the two groups. We found that 49.3% (33/67) of the tissue was benign, 44.8% (30/67) was CIS/cancerous tissue, and the remaining 4/67 samples were atypia (2) and dysplasia (2). The median AUC for the benign tissue was 19.53 (interquartile range [IQR]: 5.35-30.39) and that for CIS/cancerous tissue was 7.05 (IQR: 2.89-14.24) (P=0.002). The median wavelengths for the benign tissue and malignant tissue were 502.4nm (IQR: 500.3-504.3nm) and 505.2nm (IQR: 502.1-513.2nm), respectively (P=0.003). The median fluorescence ratio was 0.080 (IQR: 0.070-0.088) for benign tissue and 0.096 (IQR: 0.079-0.221) for CIS/cancerous tissue (P=0.002). We found statistical differences between the median AUC calculations and median wavelengths for the benign and cancerous tissue. We also found a statistical difference between the fluorescence ratios between the two tissue types. There seems to be a role for optical spectroscopy in verifying bladder lesions.

In Vivo Autofluorescence Spectroscopic Study and Evaluation of DNA Damage By Comet Assay in Smokers.

Tobacco is known environmental factor to alter the chemical composition of cells and the structure of DNA. Cellular level changes of smoker's mucosa are assessed by autofluorescence spectroscopy and the DNA damage can be evaluated by single cell gel electrophoresis (comet assay). To substantiate the changes in the autofluorescence due to smoking with that of early DNA damage without any clinical change. Group I consists of 20 individuals with normal mucosa and Group II consists of 40 individuals with smoking habit. Only males were included in this study and their age ranging from 25 to 35 years. In vivo fluorescence spectra from both groups were obtained by using hand held fiber optic probe attached to Varian Cary Eclipse fluorescence spectrophotometer and comet assay was carried out for normal and smokers by their peripheral blood. Independent-Samples t-test was used for statistical analysis. P-value was obtained to discriminate the statistical differences between the two groups. The averaged excitation and emission spectra of normal and smoker's mucosa showed significant differences statistically. In comet assay, the mean tail length of smoker group was higher than the normal group. The results showed statistically significant differences (p ≤ 0.05). These techniques will be very useful for monitoring of very early changes of mucosa before clinical manifestation of the lesion in high risk smokers and thus prevents the occurrence of premalignant disorders and early invasive carcinoma.

Optical diagnosis of the progression and reversal of CCl4-induced liver injury in rodent model using minimally invasive autofluorescence spectroscopy.

Worldwide, liver cancer is the fifth most common cancer in men and seventh most common cancer in women. Intoxicant-induced liver injury is one of the major causes for severe structural damage with fibrosis and functional derangement of the liver leading to cancer in its later stages. This report focuses on the minimally invasive autofluorescence spectroscopic (AFS) studies on intoxicant, carbon tetrachloride (CCl4)-induced liver damage in a rodent model. Different stages of liver damage, including the reversed stage, on stoppage of the intoxicant are examined. Emission from prominent fluorophores, such as collagen, nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD), and variations in redox ratio have been studied. A direct correlation between the severity of the disease and the levels of collagen and redox ratio was observed. On withdrawal of the intoxicant, a gradual reversal of the disease to normal conditions was observed as indicated by the decrease in collagen levels and redox ratio. Multivariate statistical techniques and principal component analysis followed by linear discriminant analysis (PC-LDA) were used to develop diagnostic algorithms for distinguishing different stages of the liver disease based on spectral features. The PC-LDA modeling on a minimally invasive AFS dataset yielded diagnostic sensitivities of 93%, 87% and 87% and specificities of 90%, 98% and 98% for pairwise classification among normal, fibrosis, cirrhosis and reversal conditions. We conclude that AFS along with PC-LDA algorithm has the potential for rapid and accurate minimally invasive diagnosis and detection of structural changes due to liver injury resulting from various intoxicants.

Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis.

Native fluorescence, or autofluorescence (AF), consists in the emission of light in the UV-visible, near-IR spectral range when biological substrates are excited with light at suitable wavelength. This is a well-known phenomenon, and the strict relationship of many endogenous fluorophores with morphofunctional properties of the living systems, influencing their AF emission features, offers an extremely powerful resource for directly monitoring the biological substrate condition. Starting from the last century, the technological progresses in microscopy and spectrofluorometry were convoying attention of the scientific community to this phenomenon. In the future, the interest in the autofluorescence will certainly continue. Current instrumentation and analytical procedures will likely be overcome by the unceasing progress in new devices for AF detection and data interpretation, while a progress is expected in the search and characterization of endogenous fluorophores and their roles as intrinsic biomarkers.

TIME-RESOLVED AUTOFLUORESCENCE SPECTROSCOPY AS LABEL-FREE METHOD TO CHARACTERISE ACUTE CHANGES IN EX VIVO MODELS OF CARDIAC DISEASE

Time-resolved autofluorescence spectroscopy permits characterization of biological tissues and has already shown the potential in a number of disease applications. It harnesses the photo-physical properties of a number of endogenous...

Monitoring the metabolic response to nanoencapsulated silibinin treatment in DMBA-induced oral carcinogenesis using endogenous fluorescence

Autofluorescence spectroscopy is a very sensitive tool for detecting early metabolic response after cancer treatment. The present work aims to investigate the chemopreventive effect of the prepared silibinin-loaded nanoparticles (SILNPs) relative to the efficacy of free silibinin (SIL) for monitoring the changes in the endogenous fluorophore emission and to quantify the metabolic changes in the redox state during 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch (HBP) carcinogenesis using autofluorescence spectroscopy. Significant differences in the autofluorescence spectral signatures between the control and the experimental animals have been noticed under the excitation wavelength at 320 nm with emission ranging from 350–550 nm. The tumor tissues are characterized by a decrease in the emission of collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) compared to the control tissues. Further, the optical oxidation–reduction (redox) ratio is a measure of cellular metabolism and can be determined by the relative change in the fluorescence emission intensity of NADH and FAD. The optical redox ratio will provide sufficient information on metabolic changes associated with tumor transformation. The results revealed that a significant decrease in the optical redox ratio is observed in DMBA-induced tumor tissues, which indicates increased metabolic activity compared to the control tissues. Moreover, an oral administration of SIL and its nanoparticulates restored the status of endogenous fluorophore emission and led to a higher redox ratio in the buccal mucosa of DMBA-painted animals. On a comparative basis, treatment with nanoparticulate silibinin was found to be more effective than free silibinin for reducing the formation of squamous cell carcinoma and improving the status of endogenous fluorophore emission to a near normal range in DMBA-induced hamster buccal pouch carcinogenesis. Furthermore, the diagnostic algorithms based on principal component analysis followed by linear discriminant analysis (PC–LDA) achieved an overall diagnostic sensitivity of 96.46% and specificity of 93.64% for separating the control from the experimental groups. The results of this study further suggest that the fluorescence spectroscopic technique in conjunction with PC–LDA has a potential for rapid and sensitive detection of specific metabolic alteration and changes in the endogenous fluorophores in response to anti-cancer drug treatments.

Multimodal diagnosis and visualisation of oncologic pathologies

The combined application of optical coherence tomography, Raman and autofluorescence spectroscopy of biotissues for the analysis of human malignant neoplasms is demonstrated. Rapid investigation of vast biotissue regions (at the scale of entire organs) is possible using the autofluorescence response. After selection of possible zones of pathologies one can visualise the neoplasm topology in the zone of interest with micron precision by using optical coherence tomography. In the case of suspecting the malignancy the analysis of the biotissue Raman spectrum is carried out that allows identification of the neoplasm type with the sensitivity and specificity . An experimental scheme is proposed with the combined use of the abovementioned methods, which is a prototype of the medical system for complex analysis of neoplasms.

Autofluorescence spectroscopy for NADH and flavoproteins redox state monitoring in the isolated rat heart subjected to ischemia-reperfusion

Reduction of myocardial ischemia-reperfusion injury in the patients undergoing cardiac surgery under cardiopulmonary bypass represents an important goal. Intraoperative monitoring of myocardial metabolic state using continuous registration of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) fluorescence might contribute to the solution of the problem. The successful application of fluorescent spectroscopy in the clinical field requires additional refinement of the technique, particularly using excitation of both NADH and FAD with different wavelengths. The experiments were performed on the isolated Langendorff-perfused rat hearts (n=28) subjected to either regional or global ischemia-reperfusion. Two principles of NADH and FAD autofluorescence (AF) measurement were used for ischemia monitoring: (1) analysis of photographs and videos obtained with multispectral organoscopy technique allowing the assessment of both spatial and temporal characteristics of the process (n=16); (2) continuous registration of tissue redox state in a representative area of the heart by application of local spectroscopy, assisted by fiber optic spectrometer (n=12). It was found that regional myocardial ischemia resulted in a rapid, substantial increase in the intensity of NADH AF excited at 360nm in the ischemic versus non-ischemic area of the heart. The same result was obtained when the heart was made globally ischemic, while the restoration of perfusate flow completely reversed the increase in NADH AF. During the transition from ischemia to reperfusion, the spatial heterogeneity of myocardial AF was noted on video recordings, probably reflecting the microheterogeneity of myocardial blood flow. Local spectroscopy studies demonstrated opposite changes in the NADH and FAD AF during ischemia. Using both methodological approaches, we found that repetitive brief episodes of global myocardial ischemia resulted in progressive decrease in the magnitude of AF elevation, which might point to preconditioning effect. The application of multispectral fluorescent organoscopy offers the advantage of monitoring myocardial redox state at the level of the entire heart. Local spectroscopy is characterized by better precision and, in addition, provides the unique opportunity to measure AF in different parts of the spectrum. AF measurements are non-invasive, rapid, and technically easy to perform. For future clinical applications, it might be recommended to combine the measurement of redox state of both NADH and FAD, using excitation wavelength and emission filter optimal for each fluorophore.

Comparison of Blue Wavelengths and Scan Velocity Effects on the Detection of Enamel Surface Caries Using Steady-State Laser-Induced Autofluorescence Spectroscopy

The results of sound and carious enamel fl uorescence emission study using (i) different blue wavelengths, (ii) different scan velocity, and (iii) spectral ratio are reported. The samples were irradiated using a tunable argon laser emitting at 459 and 488 nm and a 405 nm laser at two different scan velocities of 0.23 and 0.5 mm/s. The results showed a spectral band of 443–492 nm for 405 nm, 493–522 nm for 459 nm, and 526–625 nm for 488 nm lasers for sound teeth. It was found from the emission spectra that with increase in the excitation wavelength, the corresponding primary peaks of the carious samples showed Stokes shifts of 4, 6, and 2 nm, respectively. No signifi cant change was observed for the secondary peaks. Also, in all cases, the intensity of fl uorescence signals of sound teeth was higher than those of carious teeth. The highest shape factor of 1.82 and integrated intensity ratio of 1.20 were achieved at 405 nm, which provides relatively better tissue discrimination. Also, increasing the scan velocity reduced the signal amplitudes in both sound and carious samples.

623 Laser-induced autofluorescence spectroscopy: Can it be of importance in detection of bladder lesions?

623 Laser-induced autofluorescence spectroscopy: Can it be of importance in detection of bladder lesions?

Monitoring of Tumor Response to Cisplatin Using Optical Spectroscopy

INTRODUCTION: Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy–autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS: Brca1−/−; p53−/− mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS: Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS: This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response.

Fluorescence spectroscopy to discriminate neoplastic human brain lesions: a study using the spectral intensity ratio and multivariate linear discriminant analysis

Fluorescence spectroscopy is an emerging tool used to differentiate normal and malignant tissue based on the emission spectral profile from endogenous fluorophores. The goal of this study is to estimate the concentration of fluorophores using autofluorescence spectroscopy and try to utilize its diagnostic potential on samples of clinical importance. Brain tumor tissues from patients who received craniotomy for the removal of astrocytoma, glioma, meningioma and schwannoma were utilized in this study. Fluorescence emissions of the formalin fixed samples were recorded at excitation wavelengths of 320 and 410 nm. The emission characteristics of fluorophores such as collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), phospholipids and porphyrins of tumor tissue and adjacent normal tissue were elicited. Exact tissue classification was carried out using the spectral intensity ratio (SIR) and multivariate principal component analysis–linear discriminant analysis (PCA–LDA). The diagnostic algorithm based on PCA–LDA provided better classification efficiency than SIR. Moreover, the spectral data based on an excitation wavelength of 410 nm are found to be more efficient in the classification than 320 nm excitation, using PCA–LDA. Better efficacy of PCA–LDA in tissue classification was further confirmed by the receiver operator characteristic (ROC) curve method. The results of this study establish the feasibility of using fluorescence spectroscopy based real time tools for the discrimination of brain tumors from the adjacent normal tissue during craniotomies, which at present faces a huge challenge.

Optimized endoscopic autofluorescence spectroscopy for the identification of premalignant lesions in Barrett’s oesophagus

Fluorescence spectroscopy has the potential to detect early cellular changes in Barrett's oesophagus before these become visible. As the technique is based on varying concentrations of intrinsic fluorophores, each with its own optimal excitation wavelength, it is important to assess the optimal excitation wavelength(s) for identification of premalignant lesions in patients with Barrett's oesophagus. The endoscopic spectroscopy system used contained five (ultra)violet light sources (λexc=369-416 nm) to generate autofluorescence during routine endoscopic surveillance. Autofluorescence spectroscopy was followed by a biopsy for histological assessment and spectra correlation. Three intensity ratios (r1, r2, r3) were calculated by dividing the area, A, under the spectral curve of selected emission wavelength ranges for each spectrum generated by each excitation wavelength λexc as follows (Equation is included in full-text article.). Double intensity ratios were calculated using two excitation wavelengths. Fifty-eight tissue areas from 22 patients were used for autofluorescence spectra analysis. Excitation with 395, 405 or 410 nm showed a significant (P≤0.0006) differentiation between intestinal metaplasia and grouped high-grade dysplasia/early carcinoma for intensity ratios r2 and r3. A sensitivity of 80.0% and specificity of 89.5% with an area under the ROC curve of 0.85 was achieved using 395 nm excitation and intensity ratio r3. Double excitation showed no additional value over single excitation. The combination of 395 nm excitation and intensity ratio r3 showed optimal conditions to discriminate nondysplastic from early neoplasia in Barrett's oesophagus.

Fluorescence spectroscopic characterization of salivary metabolites of oral cancer patients

A pilot study has been carried out using human saliva in differentiating the normal subjects from that of oral squamous cell carcinoma (OSCC) patients, using the autofluorescence spectroscopy at 405nm excitation. A markable difference in the spectral signatures between the saliva of normal subjects and that of oral cancer patients has been noticed. The possible reasons for the altered spectral signature may be due to the presence of endogenous porphyrin, NAD(P)H and FAD in the exfoliated cells from saliva. The elevated level of porphyrin in saliva of OSCC patients may be attributed to the disturbances in the amino acid degradation pathway and heme biosynthetic pathway, during the transformation of normal into malignant cells. The integrated area under the curve of fluorescence emission spectrum at 405nm excitation and also fluorescence excitation spectrum for 625nm emission were compared for the saliva of normal and oral cancer patients. The area under the curve for the emission spectrum provides 85.7% sensitivity and 93.3% specificity, where as the fluorescence excitation spectrum discriminates the same with 84.1% sensitivity and 93.2% specificity.