Fluorescence spectroscopy and confocal fluorescence microscopy of colon benign and malignant lesions - comparative study

摘要

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.