title: Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.
abstract: 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 pO2, 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+]oand pO2changes as compared to continuous illumination.
authors.affiliation: Neuroscience Research Center, Charité Universitätsmedizin, Berlin.
authors.forename: J
authors.initials: J
authors.lastname: Rösner
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Charité Universitätsmedizin, Berlin.
authors.forename: A
authors.initials: A
authors.lastname: Liotta
authors.affiliation: Neuroscience Research Center, Charité Universitätsmedizin, Berlin.
authors.forename: E A
authors.initials: EA
authors.lastname: Angamo
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Charité Universitätsmedizin, Berlin.
authors.forename: C
authors.initials: C
authors.lastname: Spies
authors.affiliation: Neuroscience Research Center, Charité Universitätsmedizin, Berlin.
authors.forename: U
authors.initials: U
authors.lastname: Heinemann
authors.affiliation: Institute for Neurophysiology, Charité Universitätsmedizin, Berlin. richard.kovacs@charite.de.
authors.forename: R
authors.initials: R
authors.lastname: Kovács
mesh_terms: D000818:Animals; D004734:Energy Metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005453:Fluorescence; D008029:Lighting; D008297:Male; D038761:Photobleaching; D051381:Rats; D017208:Rats, Wistar
chemical_list: D005182:Flavin-Adenine Dinucleotide
keywords: Brain slice; energy metabolism; evoked field potentials; flavin adenine dinucleotide; hippocampus; photobleaching