Rösner J, Liotta A, Angamo E A, Spies C, Heinemann U, Kovács R
Neuroscience Research Center, Charité Universitätsmedizin, Berlin.
Department of Anesthesiology and Intensive Care Medicine, Charité Universitätsmedizin, Berlin.
J Microsc. 2016 Nov;264(2):215-223. doi: 10.1111/jmi.12436. Epub 2016 Jul 1.
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 , field potentials and changes in extracellular potassium concentration ([K ] ). 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 ] and pO changes as compared to continuous illumination.
黄素腺嘌呤二核苷酸（FAD）荧光的动态变化有助于深入了解线粒体内氧化还原电位的能量代谢依赖性变化。光漂白会阻碍对活组织中FAD荧光的监测，从而限制了显微荧光记录的时长。此外，这些重要电子载体的光分解会对生物样本的能量代谢和活力产生负面影响。利用脉冲发光二极管照明，我们在此确定了最佳激发设置，该设置能在海马脑片中以最低的光漂白和对代谢的干扰产生最大的荧光产量。通过监测组织氧分压、场电位和细胞外钾离子浓度（[K⁺]）的变化，研究了FAD漂白对能量代谢和活力的影响。连续照明引起的光漂白包括初始的指数下降，随后是近乎线性的衰减。脉冲照明使指数衰减明显减缓。5毫秒的脉冲长度足以达到与连续照明相当的荧光输出，而进一步增加持续时间则会增加光漂白。同样，光漂白会随着脉冲间隔的缩短而增加。光漂白具有部分可逆性，表明存在一种瞬态非荧光黄素衍生物。与连续照明相比，脉冲照明减少了FAD的光分解，提高了脑片活力以及刺激诱导的FAD、场电位、[K⁺]和氧分压变化的可重复性。
