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Title: Influence of tissue fluorescence measurement and imaging by autofluorescence of substrata
Authors: Santhosh Kumar, B
Sandhyamani, S
Nazeer, SS
Jayasree, RS
Keywords: auto-fl uorescence, substratum, photobleaching, blue shift.
Issue Date: Aug-2015
Publisher: Journal of Applied Spectroscopy
Citation: Santhosh Kumar B, Sandhyamani S, Nazeer SS, Jayasree RS. Influence of tissue fluorescence measurement and imaging by autofluorescence of substrata. Journal of Applied Spectroscopy. 2015;82(3):494-501
Abstract: Polymers, glass and fi ber reinforced materials are increasingly being used in photophysical devices. The inherent fl uorescence of devices made of these materials may itself become important during fl uorescence detection. It may interfere with or even negate the low fl uorescence signals emitted from fl uorophores molecules of interest especially when the fl uorophores exist in microvolume samples. Such effects are signifi cant when tissue micro-sections placed on substrata of high background fl uorescence. Using different excitation wavelengths, fl uorescence spectral and imaging studies were carried out to examine the effect of background fl uorescence of microslides made of various substrata such as glass, polycarbonate and aluminium on autofl uorescence of tissue sections. With decreasing wavelength of excitation, all substrata showed increased autofl uorescence. Glass showed least inherent fl uorescence and aluminium showed more refl ectance than autofl uorescence. After continuous exposure to blue and UV light, all substrata showed irreversible decreased autofl uorescence. During spectrofl uorimetric studies, a defi nite blue shift was observed in the autofl uorescence of tissue sections placed on microslides made of each of the materials. This suggests an interference of tissue autofl uorescence by background fl uorescence emitted by the substratum. This may lead to false positive or negative reporting of the fl uorophores, particularly important while analyzing micro-sections of biological samples. Microscopic imaging did not show any background fl uorescence for glass substratum with blue light. However, with UV light, glass also showed background fl uorescence during imaging. Other substrata showed strong background fl uorescence or refl ectance with both blue and UV light. This study has the potential for accurate quantifi cation of fl uorescence spectral and decay and improved fl uorescence imaging using the photobleaching effect.
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