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Micro-spectroscopy was performed with confocal laser scanning microscopy system using 405 nm excitation for fluorescence images of tissue pathologies. We had used a Leica TCS SP system (Leica Inc.). Images were acquired using a 40x magnification objective with a numerical aperture of 0.75. In Lambda-scan regime the spectral data about the selected areas of normal and abnormal tissue sites were obtained and compared. A spectral and image detection in the range of 420-680 nm had place. Fixed tissue samples are unstained, deparaffinized and rehydrated histology tissue slides. Comparison between the evaluated optical macro and micro spectroscopy parameters and their value for clinical application of autofluorescence spectroscopy in cancer diagnosis are presented and discussed.
We demonstrate the applicability of near-infrared (NIR) autofluorescence (AF) of skin tissues to differentiating neoplasms based on performing a series of experiments with
The investigated samples were cutaneous tumours ex vivo, obtained after surgical removal and kept in a formalin solution and histological sections from biopsy tissue samples, which were routinely processed for histological analysis. Comparative spectral data for benign, dysplastic nevi and pigmented malignant melanoma lesions, as well as for nonmelanoma skin tumour – basal cell carcinoma, squamous cell carcinoma and benign non-melanin pigmented pathologies – heamangioma and seboreic veruca are presented in the current report.
Fluorescence spectra obtained reveal statistically significant differences between the different benign, dysplastic and malignant lesions by the level of emission intensity, as well by spectral shape, which are fingerprints applicable for differentiation algorithms. In reflectance and absorption modes the most significant differences are related to the influence of skin pigments – melanin and hemoglobin, less pronounced is the influence of structural proteins, such as collagen and keratin. Transmission spectroscopy mode gives complementary optical properties information about the tissue samples investigated to that one of reflectance and absorption spectroscopy.
Different mechanisms solely and in combination for evaluation of the joint impact of bioenvironmental factors (stress, Helicobacter pillory, exo-toxins in the food, water, soil and air) were applied to induce gastrointestinal tract (GIT) neoplasia in rats. The transformation of damaged areas of the stomach mucosa into malignancies in all parts of gastrointestinal tract were detected using exogenous fluorescence of photosensitizers - 5-aminolevulinic acid (5-ALA) and aluminum phthalocyanine (Al-Pc). Fluorescent mapping of different organs (liver, spleen, lungs, brain) also was developed – to evaluate the distribution of the photosensitizers in the whole body on the second hour after photosensitizer application by intravenous injection. Fiber-optic probe was used to measure the organs investigated. Fluorescence spectra were detected by microspectrometer USB4000 (OceanOptics Inc., USA), and FS405 LED source on 405 nm was used as excitation source for both types of photosensitizers applied.
Diagnostically-important parameters of oximetry, optical coherence tomography and speckle-imaging of the microcirculation of the stomach were also evaluated, to evaluate changes in the blood flow and vascular architecture, during the formation of the initial phases of the neoplasm development.
Fluorescence spectra obtained reveal statistically significant differences between the different benign, dysplastic and malignant lesions by the level of emission intensity, as well by spectral shape, which are fingerprints applicable for differentiation algorithms. In reflectance mode the most significant differences are related to the influence of skin pigments – melanin and hemoglobin. Transmission spectroscopy mode gave complementary optical properties information about the tissue samples investigated to that one of reflectance and absorption spectroscopy.
Using autofluorescence detection of skin lesions we obtain very good diagnostic performance for distinguishing of nonmelanoma lesions. Using diffuse reflectance and transmission spectroscopy we obtain significant tool for pigmented pathologies differentiation, but it is a tool with moderate sensitivity for non-melanoma lesions detection. One could rapidly increase the diagnostic accuracy of the received combined “optical biopsy” method when several spectral detection techniques are applied in common algorithm for lesions’ differentiation.
Specific spectral features observed in each type of lesion investigated on micro and macro level would be presented and discussed. Correlation between the spectral data received and the microscopic features observed would be discussed in the report.
Synchronous fluorescence spectroscopy (SFS) is a steady-state approach that is used for evaluation of specific fluorescence characteristics of cancerous colorectal tissues in our studies. The feasibility of polarization fluorescence technique to enhance the contrast between normal and cancerous tissues was investigated as well. Additional linear polarizing optics was used on the way of the excitation and emission fluorescence light beams. The polarizing effects were investigated in parallel and perpendicular linear polarization modes respectively. The excitation applied was in the region of 280 – 440 nm, with 10 nm scanning step, and the fluorescence emission was detected in the region of 300 – 800 nm.
Our previous experience with SFS technique showed its great potential for accurate, highly sensitive and specific discrimination between cancerous and normal colorectal tissue. Since one of the major sources of endogenous fluorescence with diagnostic meaning is the structural protein — collagen, which is characterized with high anisotropy, we’ve expected and observed an enhancement of the spectral differences between cancerous and normal colorectal tissue, which could be beneficial for the colorectal tumour’ diagnostics using SFS.
It is important to develop and combine novel diagnostic techniques for an accurate early stage diagnosis to improve the chances for skin and GIT tumours treatment. Optical techniques are very promising methods for such noninvasive diagnosis of skin and mucosa tumours, possessing the advantages of deep imaging depth, high resolution, fast imaging speed, and noninvasive character of detection. In this study we combine autofluorescence spectroscopy and optical imaging techniques to develop more precise evaluation of the tissue pathologies investigated.
We obtain chromophore maps for GIT and cutaneous samples, with better visualization of the tumours borders and margins. In addition, fluorescence spectra give us information about the early changes in chromophores’ contents into the tissues during neoplasia growth.
In this study we present an investigation using nonlinear microscopy techniques for histological sections from biopsy tissue samples analysis. The samples were routinely processed for histological analysis and during the standard sampling the tissue slices were stained with hematoxylin and eosin dyes.
The application of nonlinear microscopy techniques, such as two photon excitation fluorescence microscopy and second harmonic generation microscopy in biomedical research for cancer diagnosis has been vastly expanding in the last few years. Two-photon excitation fluorescence microscopy is based on a non-linear optical effect of simultaneously absorption of two photons, thus achieves excited state of the absorbing molecule with energy corresponding to the sum of the energies of two incident photons. This method allows for using an excitation wavelength which is double the typically required one for excitation of diagnostically valuable endogenous fluorophores. This results in more efficient depth penetration of the longer wavelength light in the tissue. The second harmonic generation microscopy is based on the principle of the non-linear susceptibility in noncentrosymmetric structures; such structures in the tissue are formed mainly by the collagen fibers. After excitation with near-infrared photons with wavelength λ of the collagen structures, photons with wavelength ½ λ are emitted — this corresponding to the second harmonic of the excitation beam’s frequency.
The applied nonlinear microscopy techniques are suitable for detection and quantification of the morphological changes associated with stroma and epithelial transformation in colon cancer, providing complementary information about the tissue microstructure and displaying distinctive patterns between normal and malignant human colon tissues.
In this study spectral reflectance and chlorophyll fluorescence were used for the identification of biotic stress caused by the pox virus on plum trees. The spectral responses of healthy and infected leaves from cultivars, which are widespread in Bulgaria were investigated. The two applied techniques revealed statistically significant differences between the spectral data of healthy plum leaves and those infected by PPV in the visible and near-infrared spectral ranges. Their application for biotic stress detection helps in monitoring diseases in plants using the different plant spectral properties in these spectral ranges. The strong relationship between the results indicates the applicability of diffuse reflectance and fluorescence techniques for conducting health condition assessments of vegetation and their importance for plant protection practices.
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