Bremer Daniel, Leben Ruth, Mothes Ronja, Radbruch Helena, Niesner Raluca
Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany.
Neuropathology Department, Charité - Universitätsmedizin, Berlin, Berlin, Germany.
Curr Protoc Cytom. 2017 Apr 3;80:9.52.1-9.52.14. doi: 10.1002/cpcy.20.
Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.
荧光寿命成像显微镜（FLIM）是一种生成图像的技术，其中对比度是通过荧光分子的激发态寿命而非其强度和发射光谱获得的。细胞中普遍存在的辅酶烟酰胺腺嘌呤二核苷酸（NADH）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）（以下简称NAD(P)H）在游离状态下荧光寿命较短，约为400皮秒，而与酶结合时荧光寿命较长。处于这种状态的NAD(P)H的荧光寿命取决于特定酶上的结合位点。与通常在细胞中具有活性的酶相比，我们测量到与NADPH氧化酶家族成员结合的NADPH的荧光寿命为3650皮秒，而对于通常在细胞中具有活性的酶，其荧光寿命约为2000皮秒。在此，我们提出了一种基于分离细胞中NAD(P)H荧光寿命成像的可靠方案，以区分正常活性酶和主要负责氧化应激的NADPH氧化酶。© 2017约翰威立国际出版公司
