Laser-induced Autofluorescence Research Articles

POSSIBILITIES OF DETERMINING THE TIME SINCE DEATH USING THE METHODS OF MULTIDIMENSIONAL POLARIZATION AND AUTOFLUORESCENCE MICROSCOPY OF THE HUMAN VITREOUS BODY

Given the current state of affairs in practical forensic medicine and the considerable amount of scientific research on the exact determination of the postmortem interval, we conclude that the determination of the time since death is a key problem, but not yet sufficiently solved. Accurately estimating the time since death is critical to many investigations and remains one of the most challenging variables.
 Carrying out an accurate determination of the time of death is important using a complex approach, since solving this problem requires taking into account all aspects of thanatogenesis, the influence of environmental factors and other circumstances of the case. This leads to an increase in the popularity of global research involving the latest advances in medicine, chemistry, physics and forensics. The use of comprehensive research allows us to come closer to establishing the approximate range of the time since death.
 Aim of the work. Development of a complex of objective forensic medical criteria to improve the possibilities of accurately determining the time since death over a long period of time by using multidimensional polarization and autofluorescence microscopy of the human vitreous body.
 Materials and methods. The object of the study were samples of human vitreous body, deceased from cardiovascular pathology, with a known time of death.
 The following research methods were used: Mueller-matrix mapping, microscopic polarization tomography, spectral-selective laser-induced autofluorescence, statistical and wavelet analysis of the results of experimental data.
 Results. The significant effectiveness of the proposed methods in accurately determining the time of death has been demonstrated. In particular, polarization microscopic tomography using scale-selective wavelet analysis of distributions of linear birefringence of human vitreousbody layers provides a diagnostic range for determining the time since death of 36 hours with an accuracy of up to 14-16 minutes, which was not achieved by any of the existing laser polarization methods. At the same time, the combination of polarization tomography of biological tissues with spectral-selective laser fluorescence microscopy – one of the most sensitive methods,due to the assessment of the concentration parameters of molecules, opens a new window of possibilities and establishing the time since death and diagnosis of various pathological conditions. The obtained results confirm the effectiveness of this technique and show the diagnostic range of determining the time since death of 36 hours with an accuracy of up to 15-20 minutes.
 Conclusions. The innovative approach proposed in the scientific study makes a significant contribution to the development of forensic medical examination, providing more accurate and reliable identification of the time since death, which can be useful in criminal investigations and in solving other forensic tasks.

Identification of Commercial Antimalarial Herbal Drugs Using Laser-Induced Autofluorescence Technique and Multivariate Algorithms.

In malaria-prone developing countries the integrity of Anti-Malarial Herbal Drugs (AMHDs) which are easily preferred for treatment can be compromised. Currently, existing techniques for identifying AMHDs are destructive. We report on the use of non-destructive and sensitive technique, Laser-Induced-Autofluorescence (LIAF) in combination with multivariate algorithms for identification of AMHDs. The LIAF spectra were recorded from commercially prepared decoction AMHDs purchased from accredited pharmacy shop in Ghana. Deconvolution of the LIAF spectra revealed secondary metabolites belonging to derivatives of alkaloids and classes of phenolic compounds of the AMHDs. Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) were able to discriminate the AMHDs base on their physicochemical properties. Based on two principal components, the PCA- QDA (Quadratic Discriminant Analysis), PCA-LDA (Linear Discriminant Analysis), PCA-SVM (Support Vector Machine) and PCA-KNN (K-Nearest Neighbour) models were developed with an accuracy performance of 99.0, 99.7, 100.0, and 100%, respectively, in identifying AMHDs. PCA-SVM and PCA-KNN provided the best classification and stability performance. The LIAF technique in combination with multivariate techniques may offer a non-destructive and viable tool for AMHDs identification.

Automatic Classification of Antimalarial Herbal Drugs Exposed to Ultraviolet Radiation from Unexposed Ones Using Laser-Induced Autofluorescence with Chemometric Techniques.

Exposure of antimalarial herbal drugs (AMHDs) to ultraviolet radiation (UVR) affects the potency and integrity of the AMHDs. Instant classification of the AMHDs exposed to UVR (UVR-AMHDs) from unexposed ones (Non-UVR-AMHDs) would be beneficial for public health safety, especially in warm regions. For the first time, this work combined laser-induced autofluorescence (LIAF) with chemometric techniques to classify UVR-AMHDs from Non-UVR-AMHDs. LIAF spectra data were recorded from 200 ml of each of the UVR-AMHDs and Non-UVR-AMHDs. To extract useful data from the spectra fingerprint, principal components (PCs) analysis was used. The performance of five chemometric algorithms: random forest (RF), neural network (NN), support vector machine (SVM), linear discriminant analysis (LDA), and k-nearest neighbour (KNN), were compared after optimization by validation. The chemometric algorithms showed that KNN, SVM, NN, and RF were superior with a classification accuracy of 100% for UVR-AMHDs while LDA had a classification accuracy of 98.8% after standardization of the spectra data and was used as an input variable for the model. Meanwhile, a classification accuracy of 100% was obtained for KNN, LDA, SVM, and NN when the raw spectra data was used as input except for RF for which a classification accuracy of 99.9% was obtained. Classification accuracy above 99.74 ± 0.26% at 3 PCs in both the training and testing sets were obtained from the chemometric models. The results showed that the LIAF, combined with the chemometric techniques, can be used to classify UVR-AMHDs from Non-UVR-AMHDs for consumer confidence in malaria-prone regions. The technique offers a non-destructive, rapid, and viable tool for identifying UVR-AMHDs in resource-poor countries.

A Feasibility Study on Monitoring Shelf Life of Bottled Natural Fruit Juice Using Laser-Induced Autofluorescence

Shelf life of bottled natural fruit juice (BNFJ) provides relevant information on quality and authenticity for consumer protection. However, existing techniques for monitoring the shelf life of BNFJ are destructive and time-consuming. We report on using laser-induced autofluorescence (LIAF) spectroscopic technique in combination with multivariate analysis for shelf life monitoring of BNFJ. The LIAF spectra data were acquired for nine (9) continuous days on three batches of BNFJ samples purchased from a certified retailer. Deconvolution of the LIAF spectra revealed underlying peaks representing constituents of the BNFJ. Principal component analysis (PCA) was able to monitor the trend in the changes of the BNFJ as it aged. Partial least square regression (PLSR) predicted the exact day from the production of the BNFJ accurately at 96.6% and 98.8% in the training and testing sets, respectively. We, therefore, propose the LIAF combined with multivariate analysis as a potential tool for nondestructive, rapid, and relatively inexpensive monitoring of the shelf life of BNFJ.

Specific Identification of Breast Tumors Based on Laser-Induced Autofluorescence Spectroscopy

Specific Identification of Breast Tumors Based on Laser-Induced Autofluorescence Spectroscopy

The informativeness of the Johns-matrix image of laser autofluorescence in blood plasma films for early diagnosis of endometriosis associated with infertility

We used Jones-matrix images of protein networks of blood plasma films obtained from laser autofluorescence and determined the sensitivity, specificity, accuracy, and prognostication of positive and negative results. Our study aimed (1) to develop and substantiate new approaches for the diagnosis of endometriosis by improving the methods of Jones-matrix mapping of laser-induced auto-fluorescence and (2) to develop statistical approaches for analyzing the distribution of values of the true component of Jones-matrix images of blood plasma. Biological preparations were performed for two groups: 35 samples from control group 1 (women with infertility of unknown origin) and 85 samples from group 2 with endometriosis (women with infertility and endometriosis). The strength of the Jones-matrix method of autofluorescence mapping of plasma proteins taken from both groups was maximal for the decisions determined based on the calculation of the statistical moment of the 4th order, for statistical moment Z4, characterizing the sharpness of the peak distribution of the polycrystalline component of the plasma film. Comparison with the similar informative data of the Jones-matrix laser autofluorescence method of histological sections of the endometrial biopsy under conditions of blind endometriosis diagnosis revealed this method of analysis highly informative. Therefore, this technique can be used in screening studies to form a risk group.

Новая технология двухэтапного ревизионного эндопротезирования тазобедренного сустава с использованием цифровой поляризационной и лазерно-индуцированной аутофлуоресцентной микроскопии и интраоперационным изготовлением спейсеров II поколения

Актуальность. Применение лазерных источников в дифференциальной диагностике септической нестабильности компонентов эндопротеза тазобедренного сустава показало, что наибольшую чувствительность, специфичность и точность демонстрирует метод спектрополяриметрии: чувствительность достигает 92–93 %, специфичность — 82–86 %, а точность — 88–89 %. При этом исследование длится 1–2 часа. В 2017 году нами были разработаны пресс-формы для интраоперационного изготовления спейсеров ІІ поколения. Целью данного исследования было улучшение результатов лечения септической нестабильности компонентов эндопротеза тазобедренного сустава путем выполнения цифровой поляризационной микроскопии синовиальной жидкости и интраоперационного изготовления и применения цементно-антибиотиковых спейсеров ІІ поколения. Материалы и методы. Дифференциальная диагностика септического или асептического характера нестабильности компонентов эндопротеза осуществлялась благодаря проведению комплекса оптико-физических и флуоресцентных исследований поликристаллической структуры синовиальной жидкости. Применение разработанной нами технологии интраоперационного изготовления спейсера с применением пресс-форм позволяет быстро изготовить во время операции спейсер нужного размера. В цемент добавляют высокие дозы антибиотиков с учетом данных антибиотикограммы конкретного пациента. Результаты. Усовершенствованная нами технология применена при хирургическом лечении перипротезной инфекции у 52 пациентов. Проведенный анализ отдаленных результатов двухэтапного ревизионного эндопротезирования тазобедренного сустава у 49 пациентов (94,2 %) показал, что эрадикация инфекции и хорошие функциональные результаты достигнуты в 89,7 % случаев. Средняя оценка у них составила 87,18 ± 6,44 балла по шкале Harris Hip Score. Определены для статистических моментов 3-го и 4-го порядков, характеризующих асимметрию и эксцесс распределений параметра оптической анизотропии поликристаллических пленок синовиальной жидкости тазобедренного сустава, диапазоны изменения величины чувствительности — 88 % ≤ Se ≤ 96 %; специфичности — 84 % ≤ Sp ≤ 92 % и сбалансированной точности поляризационного воспроизведения распределений двулучепреломления — 86 % ≤ Ас ≤ 92 %. Выводы. При лечении 52 пациентов с перипротезной инфекцией тазобедренного сустава для выполнения первого этапа ревизионного эндопротезирования нами разработана и внедрена новая технология изготовления спейсеров ІІ поколения с применением разработанных нами пресс-форм. Стоимость такого спенсера в разы меньше, чем стоимость стандартного спенсера, изготовленного на заводе. Дифференциальная диагностика септического характера нестабильности компонентов эндопротеза осуществлялась с помощью азимутально-инвариантной Мюллер-матричной микроскопии двулучепреломления поликристаллической структуры синовиальной жидкости. Эрадикация инфекции и хорошие функциональные результаты достигнуты в 89,7 % случаев. Средняя оценка по шкале Harris Hip Score в ходе контрольного осмотра составила 87,18 ± 6,44 балла.

Laser-induced autofluorescence assisted by multivariate techniques discriminates a cataractous lens from healthy lens tissues of Sprague-Dawley rats.

Laser-induced autofluorescence (LIAF), combined with multivariate techniques, has been used to discriminate a cataractous lens from healthy lens tissues. In this study, 405 nm and 445 nm were used as excitation sources to induce the autofluorescence. Results show higher autofluorescence intensity in cataractous lens tissues than in healthy ones. Cataractous lens tissues show a red shift of 0.9 nm and 1.2 nm at 405 nm and 445 nm excitations, respectively. Using principal component analysis (PCA), three principal components (PCs) gave more than 99% variability for both 405 nm and 445 nm excitation sources. Based on the three PCs, Fisher's linear discriminant model was developed. An accuracy of 100% was obtained in classifying the lens tissues using Fisher's linear discriminant analysis (FLDA). The LIAF technique assisted by PCA and FLDA may be used for objective discrimination of cataractous lens from healthy lens tissues of Sprague-Dawley rats.

Dual-band fluorosensor for discriminating non-eating from algae-eating zooplankton in aquatic environments

We present a fluorosensor for the detection of laser-induced autofluorescence of zooplankton in marine environments. The sensor uses an inexpensive 410 nm laser diode as excitation source and simultaneously measures two fluorescence bands, 500-550 nm and 675-725 nm, using two identical 16-bit linear array detectors. We show continuous measurements at 200 Hz of zooplankton swimming through a water volume illuminated by the 410 nm laser. The sensor can distinguish salmon lice (Lepeophtheirus salmonis) larvae from an algae-eating reference species (Acartia tonsa) with a sensitivity of up to 99%. The system successfully differentiates the two species using mixed-species cultures at different ratios. This work shows the potential of fluorescent pest monitoring in the salmon farming industry and paves the way for single-ended aquatic lidars.

Exploring artificial neural network combined with laser-induced auto-fluorescence technology for noninvasive in vivo upper gastrointestinal tract cancer early diagnosis

In this study, a laser-induced auto-fluorescence (LIAF) system combined with the artificial neural network (ANN) algorithm is developed for early detection of human upper gastrointestinal tract carcinoma in vivo, through investigating the LIAF spectrum characteristics of the normal mucosa layer and the changes concerning an abnormal surface. Of the 44 participating patients, 41 underwent biopsy at the abnormal surface area at endoscopy. The ANN is employed to differentiate the LIAF data obtained from the normal and carcinoma patients (according to biopsy pathology diagnosis). The LIAF spectrum between 500 and 700 nm is selected and normalized. One data point is selected every 10 nm. A feed-forward back-propagation network with 2 hidden layers is constructed and trained. To evaluate the performance of ANN, 10 normal and 10 carcinoma data sets are tested with the trained ANN. 100% of the carcinoma data are very close to −1 (desired), 80% of the normal surface is very close to 1 (desired), and 20% return values around −0.28. Previous works on this type of ANN suggested a threshold of −0.5. As a result, all normal data are successful and the carcinoma cases are accurately classified and diagnosed. In conclusion, the LIAF technology combined with ANN diagnosis is more accurate.

Laser induced autofluorescence lifetime to identify larynx squamous cell carcinoma: Short series ex vivo study

Laser induced autofluorescence (LIAF) lifetime is useful to distinguish between normal laryngeal tissues and squamous cell carcinoma (SCC) based on variations of their biochemical composition and structure alterations. LIAF was collected from samples constituted by pairs of normal and malignant tissue, which were excised from three patients. Exclusion criteria for samples harvest were: (i) macroscopic changes of normal vocal cord observed during surgery; (ii) previous surgical intervention on vocal cord, (iii) patients treated only with chemotherapy or radiotherapy for carcinoma. Inclusion conditions: men, aged 57–68, non-smokers. A pulsed laser diode excited LIAF at 375 nm and 31 MHz repetition rate; beam full-time width at half-maximum was 87 ps at an average power of 0.49 mW. Mean LIAF lifetime for normal tissues was (3.75 ± 0.49) ns and for malignant (4.37 ± 0.85) ns: it is longer in malignant than in normal tissue. Variance analysis made with Fisher's test has shown no significant difference between patients for normal tissues; the same was true for malignant. Though, when malignant tissue was compared to normal for the same patients as well as between patients, a significant difference (significance level of 5%) was evidenced. Time-resolved LIAF may allow better differentiation between normal and malignant tissues in patients diagnosed with larynx SCC.

Compatibility of CO2 laser surgery and fluorescence detection in head and neck cancer cells.

Surgical treatment of cancer requires tumor excision with emphasis on function preservation which is achieved in (early stage) laryngeal cancer by transoral carbon dioxide (CO2 ) laser surgery. Whereas conventional laser surgery is restricted by the surgeon's visual recognition of tumor tissue, new approaches based on fluorescence-guided surgery (FGS) improve the detection of the tumor and its margin. However, it is unclear whether fluorophores are compatible with high-power laser application or whether precision is compromised by laser-induced bleaching of the dye. We applied topology-controlled 3D laser resection of fluorescent tumors cell in vitro and laser-induced autofluorescence analysis ex vivo. Laser-induced bleaching of fluorescent dyes in the visible and near-infrared light spectrum (650-900 nm) ranges below the resolution range of operation microscopes. Furthermore, specific fluorescent signals in an FGS mouse model is 104 higher than laser-induced autofluorescence in mouse tissue. Laser-induced lateral photobleaching is negligible indicating a path forward for fluorescence-guided laser surgery in head and neck cancer.

Probing endogenous collagen by laser-induced autofluorescence in burn wound biopsies: A pilot study.

The focus of the current study was to interrogate the predictive potential of laser-induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He-Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.

Case series about ex vivo identification of squamous cell carcinomas by laser-induced autofluorescence and Fourier transform infrared spectroscopy.

An ex vivo case series aimed at identification of normal laryngeal tissue from laryngeal epidermoid squamous keratinized carcinoma by measuring laser-induced autofluorescence (LIAF) and Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectra is presented. The case series results were obtained for paired samples extracted from three patients (exclusion: macroscopic changes of normal vocal cord observed during surgery; surgical intervention on vocal cord, treated only with chemotherapy or radiotherapy for carcinoma; inclusion: men, aged 57-68, non-smokers). For LIAF analysis, a 375-nm picosecond pulsed laser diode with 31MHz pulse repetition rate, 100ps full-time width at half-maximum, and average power 0.49μW was used. LIAF and FTIR-ATR spectra show noticeable differences between normal and malignant tissues. LIAF spectra differed in shape of emitted band, peak position, and band relative intensity of the two kinds of samples, evidencing hypsochromic shift and mean fluorescence intensity decrease of (75.42 ± 3)% in malignant tissue with respect to the normal one. The lack of 1745cm-1 band in FTIR-ATR spectra for malignant tissues could be considered an important indicative of the presence of this kind of tissue; moreover, it resulted a greater contribution of lipids and proteins in normal tissue and of collagen in malignant tissue. Penetration depth of the evanescent wave was about 2μm at an angle of 42°. The two spectroscopic methods are complementary, are applicable for real-time measurements, and may enhance cancer detection and diagnostics. Results presented in this study evidence the potential of the two methods for future in vivo studies.

Diagnosis and staging of caries using spectral factors derived from the blue laser-induced autofluorescence spectrum

ObjectiveThe aim of this study was to identify the factors derived from the 405nm laser-induced autofluorescence (AF) spectra that could be used to diagnose and stage caries. Materials and methodsTeeth (20 teeth per stage) were classified as sound, stage II, III, and IV based on a visual and tactile inspection. The specimens were re-examined and reclassified based on micro-CT analysis. From the teeth, the AF was obtained using a 405nm laser. Three spectral factors (spectral slope at 550–600nm, area under the curve at 500–590nm, and two-peak ratio between 625 and 667nm) were derived from the AF spectra. Using these factors, the diagnosis and staging of caries were tested, and the results were compared with those of DIAGNOdent. ResultsAfter micro-CT analysis, only 13, 11, and 13 teeth were reclassified as stages II, III, and IV, respectively. The reclassified groups showed less data overlap between the stages, and the spectral slope was 40.1–74.6, 27.5–39.6, 11.1–27.4, and 1.0–9.7 for sound, stage II, III, and IV, respectively. The differentiation of stages III and IV using DIAGNOdent appeared to be difficult due to the considerable data overlap. ConclusionAmong the factors tested, the spectral slope at 550–600nm showed the best match with the caries specimens, in which their stage had been identified precisely. Clinical significanceThe 405nm laser-induced AF spectra can be applied to the diagnosis and staging of caries alone or in conjunction with conventional methods, such as visual, tactile, and X-ray inspection.

Discrimination and quantification of autofluorescence spectra of human lung cells

To study laser-induced autofluorescence spectroscopy of the human lung cell line, we evaluated the native fluorescence properties of cancer QU-DB and normal MRC-5 human lung cells during continuous exposure to 405 nm laser light. Two emission bands centered at ~470 nm and ~560 nm were observed. These peaks are most likely attributable to mitochondrial fluorescent reduced nicotinamide adenine dinucleotide and riboflavin fluorophores, respectively. This article highlights lung cell autofluorescence characterization and signal discrimination by collective investigation of different spectral features. The absolute intensity, the spectral shape factor or redox ratio, the full width of half-maximum and the full width of quarter maximum was evaluated. Moreover, the intensity ratio, the area under the peak and the area ratio as a contrast factor for normal and cancerous cells were also calculated. Among all these features it seems that the contrast factor precisely and significantly discriminates the spectral differences of normal and cancerous lung cells. On the other hand, the relative quantum yield for both cell types were found by comparing the quantum yield of an unknown compound with known fluorescein sodium as a reference solution.

Detection of epidermoid squamous-cell carcinoma by laser induced autofluorescence -preliminary results

Epidermoid squamous-cell carcinoma is by far the most common malignant tumor of larynx (95-96%), representing 1.6-2% of cancers in men and 0.2-0.4% of malignancies in females, with a world growing incidence and a slight dominance in urban areas. By exposing cells and tissues to UV light, the excitation of naturally occurring chromophores occurs in part by non-radiative deactivations, in part via fluorescence emission. Using laser induced autofluorescence (LIAF) in natural tissues not impregnated with photosensitizers as a noninvasive autofluorescence technique for both diagnosis and intraoperative assessments of laryngeal cancer we can improve the tumor malign identification in vivo. A total of three laryngeal biopsies (i.e. three pairs of tissues, each pair containing a healthy and a tumor tissue sample extracted from the same patient) were considered in this study. The samples were collected from patients previously diagnosed with stage T3 laryngeal carcinoma. Immediately after the surgery fragments of normal tissue and neoplastic tissue were collected, fragments of which later, after freezing, were sectioned in 25-30 μm thickness slices and stretched to quartz slides. All samples were subjected to controlled laser irradiation using a pulsed diode laser (λ=375nm, pulse width=87ps, frequency 31MHz) and the autofluorescence and its lifetime were collected using two optical fibers (inner diameter 400µm and 1500µm, respectively) positioned in a 45 o geometry. The signals were recorded using a spectrograph and a photo-sensor module, the output of which was fed to a digital oscilloscope. We assessed the impact of laser induced autofluorescence and autofluorescence lifetime measurements in order to identify the differences between healthy and tumoral laryngeal tissue and outlining them, in terms of differences between the laser autoinduced fluorescence averaged intensity. The results determined the usefulness of laser induced spectroscopy in the diagnosis of laryngeal squamous cell carcinoma, discriminating between the malignant and normal tissue by analyzing the differences in spectral autofluorescence intensity and autofluorescence lifetime. Acknowledgements: This work has been financed by the National Authority for Research and Innovation in the frame of Nucleus programme -contract 4N/2016 and the project number PN-II-ID-PCE-2011-3-0922. The authors thank Prof. M. L. Pascu for permanent support in performing this research.

Mueller-matrix of laser-induced autofluorescence of polycrystalline films of dried peritoneal fluid in diagnostics of endometriosis.

This research presents investigation results of the diagnostic efficiency of an azimuthally stable Mueller-matrix method of analysis of laser autofluorescence of polycrystalline films of dried uterine cavity peritoneal fluid. A model of the generalized optical anisotropy of films of dried peritoneal fluid is proposed in order to define the processes of laser autofluorescence. The influence of complex mechanisms of both phase (linear and circular birefringence) and amplitude (linear and circular dichroism) anisotropies is taken into consideration. The interconnections between the azimuthally stable Mueller-matrix elements characterizing laser autofluorescence and different mechanisms of optical anisotropy are determined. The statistical analysis of coordinate distributions of such Mueller-matrix rotation invariants is proposed. Thereupon the quantitative criteria (statistic moments of the first to the fourth order) of differentiation of polycrystalline films of dried peritoneal fluid, group 1 (healthy donors) and group 2 (uterus endometriosis patients), are determined.

Spectral characteristics of caries-related autofluorescence spectra and their use for diagnosis of caries stage.

The purpose of the present study was to identify factors useful for diagnosis of the caries stage from laser-induced autofluorescence (AF) spectra. Affected teeth were accurately staged and allocated to four groups: sound, stage II, stage III, or stage IV. A 405-nm laser was used to produce AF spectra. The spectrum factors analyzed were spectrum slope at 550 to 600 nm, spectral area from 500 and 590 nm, and intensity ratio of peaks 625 and 667 nm ( 625/667 nm ). DIAGNOdent was used as control measurement. AF spectra of sound teeth had a peak near 500 nm followed by a smooth decline to 800 nm. As caries progressed, some specimens in stages II to IV showed one or two peak(s) near 625 and 667 nm. Slopes at 550 to 600 nm and areas under the curve at 500 to 590 nm were significantly different ( p 0.001 ) for each stage. Two-peak ratios were also significantly different ( p 0.001 ) except for stage III and stage IV. DIAGNOdent readings for sound and stage II and stage III and IV were not significantly different. Among the studied factors, the spectrum slope at 550 to 600 nm and area under curve at 500 to 590 nm could be useful treatment decision-making tools for carious lesions.

Nonlinear Behavior of the Autofluorescence Intensity on the Surface of Light-Scattering Biotissues and its Theoretical Proof.

In the up-to-date medical laser fluorescence spectroscopy (LFS) in vivo, there is a problem of quantification of fluorophores concentrations in optically-turbid biotissues by measurements of the laser induced autofluorescence flux on the surface of these tissues. One of the main problems is: whether the flux depends linearly or non-linearly on the concentration of fluorophores in tissues? The purpose of this work was both experimental and theoretical study of the character of dependencies between measured fluorescence intensities and fluorophores concentrations in optically-turbid media. In the experimental part of our study, measurements of the superficial fluorescence on phantoms at various known concentrations of fluorophores in them were carried out. As a result, experimental dependencies of registered intensities of the laser-induced autofluorescence emission were plotted against fluorophore concentrations. In the theoretical part of the study, the analytical solution for the fluorescence emission by Kokhanovsky's method based on the well-known two-flux Kubelka-Munk approach (KMA) was used. In addition, in our study the Kokhanovsky's method was modified by its association with our improved KMA, allowing us to receive exact analytical solutions for boundary intensities collected by optical probes. As a result, a set of theoretical curves describing the influence of fluorophore concentrations on the registered autofluorescence intensities was obtained, as well. Both experimental and theoretical results show a good qualitative agreement with each other. Also, these results demonstrate that the dependence of the fluorescence intensity on tissues' optical properties and on the concentration of fluorophores in light-scattering tissues can be both nonlinear and non-monotonic.