Characterization and classification of ductal carcinoma tissue using four channel based stokes-mueller polarimetry and machine learning.

Interaction of polarized light with healthy and abnormal regions of tissue reveals structural information associated with its pathological condition. Even a slight variation in structural alignment can induce a change in polarization property, which can play a crucial role in the early detection of abnormal tissue morphology. We propose a transmission-based Stokes-Mueller microscope for quantitative analysis of the microstructural properties of the tissue specimen. The Stokes-Mueller based polarization microscopy provides significant structural information of tissue through various polarization parameters such as degree of polarization (DOP), degree of linear polarization (DOLP), and degree of circular polarization (DOCP), anisotropy (r) and Mueller decomposition parameters such as diattenuation, retardance and depolarization. Further, by applying a suitable image processing technique such as Machine learning (ML) output images were analysed effectively. The support vector machine image classification model achieved 95.78% validation accuracy and 94.81% testing accuracy with polarization parameter dataset. The study's findings demonstrate the potential of Stokes-Mueller polarimetry in tissue characterization and diagnosis, providing a valuable tool for biomedical applications.

Polarization-resolved Stokes-Mueller imaging: a review of technology and applications.

Polarization microscopy, a powerful optical tool to study anisotropic properties of biomolecules, provides better microstructural information of a sample as compared with conventional optical microscopic techniques. The measurement and analysis of polarization states of light can be performed using both Jones matrix as well as Stokes algebra. Further, the details of optical properties of specimen are characterized by Mueller matrix. However, the application of Jones calculus is limited to perfectly polarized light, but Stokes-Mueller polarimetry is emerging as a promising tool for tissue imaging due to its application irrespective of polarization state of the light. In this review article, we explain the development of Stokes-Mueller formalism in context of linear optics. Furthermore, application of Mueller matrix decomposition (MMD) method to derive sample properties is demonstrated in several bio-medical studies.

Laser induced autofluorescence in the monitoring of ß-mercaptoethanol mediated photo induced proton coupled electron transfer in proteins.

Photo induced proton coupled electron transfer (PCET) is an important process that many organisms use for progression of catalytic reactions leading to energy conversion. In the present study, the influence of SDS and BME on the redox properties of tyrosine and tryptophan for five different globular proteins, BSA, HSA, RNase-A, trypsin and lysozyme were studied using laser induced autofluorescence. The proteins were subjected to denaturation under SDS, SDS plus heat and SDS plus ß-mercaptoethanol (BME) plus heat and the corresponding fluorescence were recorded. The influence of BME on the autofluorescence properties of the proteins were evaluated upon tris-2-corboxy-ethyl phosphine (TCEP) denaturation. The BSA and HSA when exposed to SDS alone, exhibited hydrophobic collapse around their tryptophan moieties. However, these proteins when treated with SDS plus BME plus heat, an unusual red shift in the emission was observed, may be due to proton transfer from hydroxyl group of the excited tyrosine residues to the local microenvironments. The observation was further confirmed with similar proton transfer in absence of tryptophan in RNase-A showing involvement of tyrosine in the process. A drastic quenching of fluorescence in all of the proteins under study were also observed, may be due to photo-induced electron transfer (PET) from BME to the intrinsic fluorophores resulting in radical ions formation, evaluated upon DCFDA measurements.

Prediction of absorption coefficients by pulsed laser induced photoacoustic measurements.

In the current study, a pulsed laser induced photoacoustic spectroscopy setup was designed and developed, aiming its application in clinical diagnostics. The setup was optimized with carbon black samples in water and with various tryptophan concentrations at 281nm excitations. The sensitivity of the setup was estimated by determining minimum detectable concentration of tryptophan in water at the same excitation, and was found to be 0.035mM. The photoacoustic experiments were also performed with various tryptophan concentrations at 281nm excitation for predicting optical absorption coefficients in them and for comparing the outcomes with the spectrophotometrically-determined absorption coefficients for the same samples. Absorption coefficients for a few serum samples, obtained from some healthy female volunteers, were also determined through photoacoustic and spectrophotometric measurements at the same excitations, which showed good agreement between them, indicating its clinical implications.

Autofluorescence of osteoporotic mouse femur bones: a pilot study.

OBJECTIVE: The objective of this study was to evaluate the efficacy of laser-induced fluorescence (LIF) in combination with principal component analysis (PCA) to characterize osteoporotic conditions induced by ovariectomy (OVX) in mice. BACKGROUND DATA: The dual energy X-ray absorptiometry is the gold standard methodology routinely used to diagnose osteoporosis. In recent years, the use of LIF to characterize human disease and to aid in diagnosis has shown great promise. However, this technique has not been much exploited for monitoring osteoporosis. MATERIALS AND METHODS: Swiss albino mice were sacrificed 2, 3, 4, and 5 wk after OVX and their femur bones were excised. The same protocol was used for age-matched female controls (no OVX). The LIF spectra from different regions of the bones were recorded and compared using PCA. RESULTS: A significant change in the fluorescence pattern of osteoporotic bones compared with the control was indicated by PCA match/no-match analysis. A region-wise PCA match/no-match analysis of the spectral changes against respective region calibration sets indicated more no-matches in the fifth week bones compared with the others. Further, the spectral differences were more prominent in the proximal and distal parts of the bones. In addition to the PCA, Gaussian curve fitting was also performed on control, third week, and fifth week bone spectra to identify different spectral components in them. CONCLUSION: This preliminary study using fluorescence spectroscopy in combination with PCA clearly demonstrated osteoporotic changes in mouse femur bones at different time points after OVX, suggesting possible human applications.

Autofluorescence of breast tissues: evaluation of discriminating algorithms for diagnosis of normal, benign, and malignant conditions.

OBJECTIVE: We evaluated different discriminating algorithms for classifying laser-induced fluorescence spectra of normal, benign, and malignant breast tissues that were obtained with 325-nm excitation. BACKGROUND DATA: Mammography and histopathology are the conventional gold standard methods of screening and diagnosis of breast cancers, respectively. The former is prone to a high rate of false-positive results and poses the risk of repeated exposure to ionizing radiation, whereas the latter suffers from subjective interpretations of morphological features. Thus the development of a more reliable detection and screening methodology is of great interest to those practicing breast cancer management. Several studies have demonstrated the efficacy of optical spectroscopy in diagnosing cancer and other biomedical applications. MATERIALS AND METHODS: Autofluorescence spectra of normal, benign, and malignant breast tissues, with 325-nm excitation, were recorded. The data were subjected to diverse discriminating algorithms ranging from intensities and ratios of curve-resolved bands to principal components analysis (PCA)-derived parameters. RESULTS: Intensity plots of collagen and NADPH, two known fluorescent biomarkers, yielded accurate classification of the different tissue types. PCA was carried out on both unsupervised and supervised methods, and both approaches yielded accurate classification. In the case of the supervised classification, the developed standard sets were verified and evaluated. The limit test approach provided unambiguous and objective classification, and this method also has the advantage of being user-friendly, so untrained personnel can directly compare unknown spectra against standard sets to make diagnoses instantly, objectively, and unambiguously. CONCLUSION: The results obtained in this study further support the efficacy of 325-nm-induced autofluorescence, and demonstrate the suitability of limit test analysis as a means of objectively and unambiguously classifying breast tissues.

Autofluorescence of normal, benign, and malignant ovarian tissues: a pilot study.

OBJECTIVE: The objective of this study is to evaluate the efficacy of laser-induced fluorescence (LIF) data obtained at 325-nm pulsed laser excitation for the discrimination of normal, benign, and malignant ovarian tissues. BACKGROUND DATA: Several studies have reported that the autofluorescence technique has a high specificity and sensitivity for discrimination between diseased and non-diseased tissues of various cancers, and also has the advantages of being non-invasive and producing a real-time diagnosis. When using this technique on ovarian tissues in most of the previously reported studies, multivariate statistical tools were used and classification analyses were carried out. MATERIALS AND METHODS: Autofluorescence spectra of normal, benign, and malignant ovarian tissues were recorded with 325-nm pulsed laser excitation in the spectral region from 350-600 nm in vitro. The spectral analysis for discrimination between the different types of tissues was carried out using principal component analysis (PCA)-based non-parametric k-nearest neighbor (k-NN) analysis. RESULTS: A total of 97 (34 normal, 33 benign, and 30 malignant) spectra were obtained from 22 subjects with normal, benign, and malignant tissues. The discrimination analysis of data using a PCA-based k-NN algorithm showed very good discrimination. The performance of the analysis was evaluated by calculating statistical parameters, specificity, sensitivity, and accuracy and were found to be 100%, 90.90%, and 94.2%, respectively. CONCLUSION: The results show that the discrimination of normal, benign, and malignant ovarian conditions can be achieved quite successfully using LIF.

Dynamics of L-tryptophan in aqueous solution by simultaneous laser induced fluorescence (LIF) and photoacoustic spectroscopy (PAS).

An experimental system for measuring simultaneously photoacoustic (PA) and fluorescence signals is described. The simultaneous measurement of laser induced fluorescence and photoacoustic signals provide a suitable method for the study of different quenching phenomena occurring in fluorescent systems. In this paper we report tryptophan solvation dynamics in water using fluorescence and photoacoustic spectra recorded simultaneously by photoacoustic and fluorescence signals as functions of concentration, indicate that quantum yield is maximum at low concentrations. Also, the energy lost in the fluorescence path of tryptophan, due to different quenching phenomena like self quenching, Resonance energy transfer (RET), solvation relaxation, etc. is clearly seen from the photoacoustic signal intensity which increases as the fluorescence intensity decreases.

Optical pathology using oral tissue fluorescence spectra: classification by principal component analysis and k-means nearest neighbor analysis.

The spectral analysis and classification for discrimination of pulsed laser-induced autofluorescence spectra of pathologically certified normal, premalignant, and malignant oral tissues recorded at a 325-nm excitation are carried out using MATLAB@R6-based principal component analysis (PCA) and k-means nearest neighbor (k-NN) analysis separately on the same set of spectral data. Six features such as mean, median, maximum intensity, energy, spectral residuals, and standard deviation are extracted from each spectrum of the 60 training samples (spectra) belonging to the normal, premalignant, and malignant groups and they are used to perform PCA on the reference database. Standard calibration models of normal, premalignant, and malignant samples are made using cluster analysis. We show that a feature vector of length 6 could be reduced to three components using the PCA technique. After performing PCA on the feature space, the first three principal component (PC) scores, which contain all the diagnostic information, are retained and the remaining scores containing only noise are discarded. The new feature space is thus constructed using three PC scores only and is used as input database for the k-NN classification. Using this transformed feature space, the centroids for normal, premalignant, and malignant samples are computed and the efficient classification for different classes of oral samples is achieved. A performance evaluation of k-NN classification results is made by calculating the statistical parameters specificity, sensitivity, and accuracy and they are found to be 100, 94.5, and 96.17%, respectively.

Principal component analysis and artificial neural network analysis of oral tissue fluorescence spectra: classification of normal premalignant and malignant pathological conditions.

Pulsed laser-induced autofluorescence spectroscopic studies of pathologically certified normal, premalignant, and malignant oral tissues were carried out at 325 nm excitation. The spectral analysis and classification for discrimination among normal, premalignant, and malignant conditions were performed using principal component analysis (PCA) and artificial neural network (ANN) separately on the same set of spectral data. In case of PCA, spectral residuals, Mahalanobis distance, and scores of factors were used for discrimination among normal, premalignant, and malignant cases. In ANN, parameters like mean, spectral residual, standard deviation, and total energy were used to train the network. The ANN used in this study is a classical multiplayer feed-forward type with a back-propagation algorithm for the training of the network. The specificity and sensitivity were determined in both classification schemes. In the case of PCA, they are 100 and 92.9%, respectively, whereas for ANN they are 100 and 96.5% for the data set considered.

Optical properties of Dy3+ doped in oxyfluoroborate glass.

Optical absorption, fluorescence and photoacoustic spectra of Dy3+ doped in oxyfluoroborate glass has been studied. Lifetime of 4F(9/2) level has been measured for different concentration of Dy3+. Effect of concentration quenching on the lifetime and the fluorescence yield has been observed and the mechanism of energy transfer discussed. The energy transfer from Pr3+ to Dy3+ has been observed and explained in a codoped glass containing Pr3+ + Dy3+.

Optical studies of Eu3+ doped oxyfluoroborate glass.

Rare earth doped oxyfluoroborate glasses have been prepared with different concentration of Eu3+. The UV-Vis/NIR optical absorption, laser induced fluorescence and photoacoustic spectra of Eu3+ in this host have been studied. Different optical parameters such as oscillator strength, Judd-Ofelt intensity parameter, stimulated emission cross-section, transition probability, branching ratio and radiative lifetime, etc. have been calculated. Lifetime of the 5D0 level at various concentrations of Eu3+ have been used to explain the concentration dependent fluorescence quenching. The mechanism of quenching was found to be dipole-dipole. Energy transfer have also been studied from Eu3+ to Pr3+ in sample with 1 mol% (Eu3+) + 1 mol% Pr3+.

Osteoradionecrosis (ORN) of the mandible: a laser Raman spectroscopic study.

Laser Raman spectroscopy has been used in this study to characterize mandibular bone samples from patients who had undergone radiation therapy for oral cancer. The paper discusses spectral changes resulting in osteoradionecrosis (ORN) of the mandibular bone, a serious complication that may occur after radiation therapy. Histopathological studies normally reveal the radiation damage on vascular canals and loss in bone cells, but will not reveal any structural or biochemical changes. All radiation-induced side effects are attributed to this hypovascularity and hypocellularity caused by early- and/or late-delayed effects. Our Raman studies on normal and ORN bone and on bone exposed to radiation, but not in the ORN state, show that irradiation produces immediate structural changes in the inorganic bone matrix with a slight loss in cells. ORN bone, in addition to the structural changes that had already occurred on radiation exposure, shows almost complete loss of cellular components. Since bone tissue is continuously being remodeled (dissolved and rebuilt) under normal conditions, our results suggest that the immediate structural changes in the calcium hydroxy apatite mineral part is not repaired in ORN, due to loss of the highly transient osteoblasts and osteoclasts resulting from destruction of stem cells. The spectral studies also show changes in the organic matrix, which is mostly type I collagen.

Laser spectroscopic studies of Tb3+-doped oxyfluoroborate glass.

Tb3+-doped oxyfluoroborate glasses have been prepared for different concentrations of Tb. The absorption, fluorescence and photoacoustic spectra of these have been recorded and studied. It is marked that the fluorescence intensity of different fluorescence transitions decreases with the increase of Tb ion concentration in the glass. This quenching at higher concentration is due to the energy transfer among the excited and nearest neighbor unexcited Tb ions in the glass. The lifetime measurement confirms it, as the lifetime of a particular state was found to decrease with the increase of Tb ion concentration in the glass. The mechanism of the energy transfer process was determined to involve quadrupole quadrupole interaction. We have also studied the energy transfer from Tb3+-->Pr3+ when both the rare earths are doped together in the glass. A decrease in the lifetime of the 5D4 level of Tb3+ with the increase of Pr3+ concentration confirms this.