Krishna Kishore Mahato

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.

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.

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT)

The present study focuses on the evaluation of the effect of He-Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post-wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non-illuminated controls.

Development and evaluation of fiber optic probe-based helium-neon low-level laser therapy system for tissue regeneration--an in vivo experimental study.

We report the design and development of an optical fiber probe‐based Helium–Neon (He–Ne) low‐level laser therapy system for tissue regeneration. Full thickness excision wounds on Swiss albino mice of diameter 15 mm were exposed to various laser doses of 1, 2, 3, 4, 6, 8 and 10 J cm−2 of the system with appropriate controls, and 2 J cm−2 showing optimum healing was selected. The treatment schedule for applying the selected laser dose was also standardized by irradiating the wounds at different postwounding times (0, 24 and 48 h). The tissue regeneration potential was evaluated by monitoring the progression of wound contraction and mean wound healing time along with the hydroxyproline and glucosamine estimation on wound ground tissues. The wounds exposed to 2 J cm−2 immediately after wounding showed considerable contraction on days 5, 9, 12, 14, 16 and 19 of postirradiation compared with the controls and other treatment schedules, showing significant (P < 0.001) decrease in the healing time. A significant increase in hydroxyproline and glucosamine levels was observed for the 2 J cm−2 irradiation group compared with the controls and other treatment groups. In conclusion, the wounds treated with 2 J cm−2 immediately after the wounding show better healing compared with the controls.

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.

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.

Effect of laser dose and treatment schedule on excision wound healing in diabetic mice.

The present study was undertaken to evaluate a He‐Ne laser (632.8 nm; 7 mW; 4.02 mW cm−2; 15 mm spot size) dose and the treatment schedule on diabetic wound healing in a mouse model. Circular wounds of 15 mm diameter were created on streptozotocin induced diabetic Swiss albino mice, and were uniformly illuminated with the single exposure of various He‐Ne laser doses of 1, 2, 3, 4 and 5 J cm−2 respectively. Further, the treatment schedule was also optimized by exposing the wounds with 3 J cm−2 at 0, 24 h, 48 h postwounding. Contraction kinetics, mean area under the curve and the mean healing time of the wounds were computed along with the collagen and the glucosamine levels in the wound ground tissues at various postwounding treatment schedules. Results of this study indicated that the single exposure of 3 J cm−2 laser dose applied immediately after the wounding caused a significant reduction in the mean area under the curve and the mean healing time along with the elevated levels of collagen and glucosamine contents in the tissue compared to the controls. In conclusion, He‐Ne laser dose of 3 J cm−2 applied immediately after the wounding has demonstrated optimum wound healing compared to the other doses and treatment schedules.

Photoacoustic spectroscopy of ovarian normal, benign, and malignant tissues: a pilot study

Photoacoustic spectra of normal, benign, and malignant ovarian tissues are recorded using 325-nm pulsed laser excitation in vitro. A total of 102 (34 normal, 38 benign, and 30 malignant) spectra are obtained from 22 samples belonging to normal, benign, and malignant subjects. Applying multi-algorithm approach, comprised of methods such as, principal component analysis (PCA) based k-nearest neighbor (k-NN) analysis, artificial neural network (ANN) analysis, and support vector machine (SVM) analysis, classification of the data has been carried out. For PCA, first the calibration set is formed by pooling 45 spectra, 15 belonging to each of pathologically certified normal, benign, and malignant samples. PCA is then performed on the data matrix, comprised of the six spectral features extracted from each of 45 calibration samples, and three principal components (PCs) containing maximum diagnostic information are selected. The scores of the selected PCs are used to train the k-NN, ANN, and SVM classifiers. The ANN used is a classical multilayer feed forward network with back propagation algorithm for its training. For k-NN, the Euclidean distance based algorithm is used and for SVM, one-versus-rest multiclass kernel-radial basis function is used. The performance evaluation of the classification results are obtained by calculating statistical parameters like specificity and sensitivity. ANN and k-NN techniques showed identical performance with specificity and sensitivity values of 100 and 86.76%, whereas SVM had these values at 100 and 80.18%, respectively. In order to determine the relative diagnostic performance of the techniques, receiver operating characteristics analysis is also performed.

Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.

Influence of Helium‐Neon Laser Irradiation on Seed Germination In Vitro and Physico‐Biochemical Characters in Seedlings of Brinjal (Solanum melongena L.) var. Mattu Gulla

In the present study, the seeds of brinjal (Solanum melongena L.) var. Mattu Gulla were irradiated with single exposure of He–Ne laser at different doses of 5–40 J cm−2 and germinated aseptically. Thirty day old seedlings were harvested and the germination, growth, physiological and biochemical parameters were estimated and compared with un‐irradiated control seedlings. A significant enhancement in growth characters were noted with respect to length, fresh and dry weight of shoots and roots. In addition, chlorophyll (a and b), carotenoid content, anthocyanin and amylases (α and β) activities were found to be altered. Significant alterations in percentage of seed germination (P < 0.001) and time to 50% germination (P < 0.001) were observed in the irradiated seeds compared with the un‐irradiated controls. In conclusion, the results of the present study demonstrated that low dose (5–30 J cm−2) of He–Ne laser irradiation enhanced the germination process and altered growth, by positively influencing physiological and biochemical parameters of the brinjal seedlings compared with un‐irradiated control under in vitro conditions.