Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO(2), field potentials and changes in extracellular potassium concentration ([K+](o)). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K+](o) and pO(2) changes as compared to continuous illumination. Lay description Neuronal activity is accompanied by changes in neuronal and glial energy metabolism. Redox state dependent changes in flavin adenine dinucleotide (FAD) fluorescence report on alterations of intracellular energy metabolism. However, its use in biological probes is limited by the strong photobleaching observed with fluorescence microscopy. Here we described the dependence of FAD photobleaching on the frequency and duration of short LED pulses and compared the effects of continuous versus pulsed illumination on evoked FAD, pO2, extracellular potassium and field potential transients in hippocampal brain slices. While continuous FAD-excitation induced multiexponential photobleaching resulted in disturbances in energy metabolism, pulsed LED illumination yielded the same fluorescence output without affecting brain slice viability.