Influence of Tissue Fluorescence Measurement and Imaging by Auto-Fluorescence of Substrata

Polymers, glass and fiber reinforced materials are increasingly being used in photophysical devices. The inherent fluorescence of devices made of these materials may itself become important during fluorescence detection. It may interfere with or even negate the low fluorescence signals emitted from fluorophores molecules of interest especially when the fluorophores exist in microvolume samples. Such effects are significant when tissue micro-sections placed on substrata of high background fluorescence. Using different excitation wavelengths, fluorescence spectral and imaging studies were carried out to examine the effect of background fluorescence of microslides made of various substrata such as glass, polycarbonate and aluminium on autofluorescence of tissue sections. With decreasing wavelength of excitation, all substrata showed increased autofluorescence. Glass showed least inherent fluorescence and aluminium showed more reflectance than autofluorescence. After continuous exposure to blue and UV light, all substrata showed irreversible decreased autofluorescence. During spectrofluorimetric studies, a definite blue shift was observed in the autofluorescence of tissue sections placed on microslides made of each of the materials. This suggests an interference of tissue autofluorescence by background fluorescence emitted by the substratum. This may lead to false positive or negative reporting of the fluorophores, particularly important while analyzing micro-sections of biological samples. Microscopic imaging did not show any background fluorescence for glass substratum with blue light. However, with UV light, glass also showed background fluorescence during imaging. Other substrata showed strong background fluorescence or reflectance with both blue and UV light. This study has the potential for accurate quantification of fluorescence spectral and decay and improved fluorescence imaging using the photobleaching effect.