Neuroscience Research Center, Charité-Universitätsmedizin Berlin, Germany.
J Neurosci Methods. 2013 Jan 30;212(2):222-7. doi: 10.1016/j.jneumeth.2012.11.001. Epub 2012 Nov 8.
Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence.
烟酰胺腺嘌呤二核苷酸（NAD(P)H）和黄素腺嘌呤二核苷酸（FADH₂）是参与细胞能量代谢和多种酶促过程的电子载体。由于还原型 NAD(P)H 和氧化型 FAD 分子具有荧光性，组织自发荧光的变化为细胞氧化还原状态和能量代谢提供了有价值的信息。由于汞弧灯（HBO）的荧光激发本质上与光漂白和光毒性相关，因此，通过更换 HBO 灯为发光二极管（LED）可能会使微荧光监测能量代谢受益。在这里，我们描述了一个基于 LED 的定制设置，用于监测单个细胞（HEK293）和脑片的 NAD(P)H 和 FAD 荧光。我们比较了两种光源（HBO 灯和 LED）的 NAD(P)H 漂白特性，以及三种不同探测器类型（多像素光子计数器（MPPC）、光电倍增管（PMT）和光电二极管）的灵敏度和信噪比。与 HBO 灯激发相比，LED 激发在相同的荧光输出下导致光漂白减少。暂时增加 LED 功率会导致 NAD(P)H 荧光可逆漂白。恢复动力学取决于代谢底物，表明 NAD(P)H 荧光与代谢的偶联。脑片的电刺激会引起 NAD(P)H/FAD 荧光瞬变的双相氧化还原变化。增加 PMT 的增益并降低 LED 功率可获得与 MPPC 和光电二极管相同的灵敏度，而不会降低信号噪声比。总之，用 LED 取代 HBO 灯可能会改善传统的基于 PMT 的组织自发荧光微荧光法。
