Endogenous fluorescence detection of neoplastic tissues could become a sensitive add-in tool for early and precise detection of different tumours in soft tissues. Endoscopic techniques and fiber-optical fluorescent probes allow achieving all organs and places in human body. New light excitation sources and sensitive detectors with high spectral resolution allow detecting low-intensity autofluorescence or to distinguish signals of specific fluorescent markers accumulated in the tumor cells. The most important moment in the current development of steady-state fluorescence technique for diagnostic applications in oncological practice is to develop appropriate databases with information about fluorescent properties of different types of pathologies for each organ and biological tissue types. One need to estimate the influence of cellular environment and state, presence of different intrinsic chromophores and pigments that could distort the emission signals detected. From diagnostic point of view, information about different tumour sub-types, state of growth, dysplastic and benign forms of lesions has to be accumulated and used for differential analysis during primary clinical observations. In this report will be presented experience on endogenous fluorescent detection based on excitation-emission matrix (EEM) development and synchronous fluorescence spectroscopy (SFS) of lower part gastrointestinal tract tumours. Malignant carcinoma lesions of colon and rectum were detected and differentiated from the normal mucosa in broad spectral range (excitation at 280-440 nm, emission at 300-800 nm) by the autofluorescent properties revealed.
Macroscopic fluorescence spectroscopy parameters of colon benign and malignant tissues were obtained with spectrofluorimeter FluoroLog 3 (HORIBA Jobin Yvon, France). Investigated biological tissue samples were excised during standard surgical procedure for tumour removal, with cancerous and healthy tissue and the measurements are performed as soon as possible after the excision. The tissue samples were stored in safe-keeping perfusion solution and isothermal conditions and transported from the hospital to the spectroscopy laboratory. The fluorescence spectroscopy measurements were performed for excitation wavelengths in the range of 280-440 nm, with 10 nm increment step, and emission was detected in the range of 300-800 nm for the healthy and cancerous areas of the sample with a step of 1 nm.
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.
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