Corneal respiratory function by FAD autofluorescence lifetime

Abstract

AbstractPurpose We intend to develop an efficient method to assess in‐vivo the corneal respiratory function in order to diagnose corneal cells dysfunction prior to its pathologic expression.Methods Metabolic alterations can be assessed by measuring the amount of the metabolic co‐factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). FAD has advantages over NADH, like being present only in the mitochondria. Furthermore, using fluorescence lifetime imaging microscopy (FLIM) we are able to discriminate between its free or protein‐bound states. We resorted to a PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan). This setup uses a pulsed blue diode laser with a trigger frequency of 40 MHz with an excitation filter of 440±10 nm. Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH). The instrument response function was acquired to improve data analysis precision.Results We successfully acquired ex‐vivo autofluorescence images of male Wistar rat and Bovine corneas. A bi‐exponential decay was observed in both cases with a fast decay around 1 ns and a longer one around 4 ns, which correspond to protein‐bound and free FAD, respectively. These results are in accordance with other studies, although there is some controversy regarding FAD lifetimes.Conclusion We showed that it is possible, with our apparatus, to acquire metabolic images of the cornea using FAD autofluorescence. We intend to modify the instrument optical setup in order to acquire reflectance and fluorescence lifetime images simultaneously for corneal layer identification.