Investigation of intra‐cellular metabolism by time‐resolved autofluorescence

Abstract

AbstractPurpose Changes in metabolism are first signs of pathological processes. The extra‐cellular metabolism can be estimated by parameters of microcirculation. The fluorescence of endogenous fluorophores characterizes the intracellular metabolism. Besides of NADH and FAD which act as electron transporter in the respiratory chain, several other fluorophores like lipofuscin and advanced glycation end products, or collagen and elastin form the autofluorescence of the fundus.Methods The influence of single fluorophores can be determined combining measurements of fluorescence decay after pulse excitation with spectral measurements. Based on fluorescence and lifetime measurements on isolated fluorophores and separated anatomical structures of porcine eyes, a laser scanner ophthalmoscope was modified for Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO). The eye‐ground of human eyes is excited at 448 nm (70 ps FWHM, 80 MHz) and the fluorescence is detected in 2 spectral channels (490‐560 nm and 560‐700 nm) in TCSPC in 1024 time channels.Results A certain relation was found between exponents of triple‐exponential fit and anatomical structures. The lifetime tau 1 (about 70 ps) corresponds to the fluorescence in retinal pigment epithelium. The lifetime tau 2 (about 400 ps) originates from layers in the neuronal retina (receptors, bi‐polares, ganglion cells). The lifetime tau 3 (about 3ns) is determined by connective tissue in the nerve fiber layer and to a certain degree by the fluorescence of the crystalline lens, too.Conclusion The changes of tau 2 in the short wavelength spectral range which were found in the neuronal retina for diabetic patients having no signs of diabetic retinopathy,are interpretable as a loss of free NADH and a shift to protein‐bound NADH. Commercial interest