Core Facility Confocal and Multiphoton Microscopy, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Laser-Forschungslabor, LIFE Center, University Hospital, LMU Munich, Fraunhoferstrasse 20, 82152 Planegg, Germany.
Int J Mol Sci. 2021 May 31;22(11):5952. doi: 10.3390/ijms22115952.
Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.
代谢型 FLIM（荧光寿命成像）用于对细胞和组织中的生物能量状态进行成像。虽然辅酶的荧光寿命被归因于细胞代谢的指示剂已被广泛接受，但对于细胞的氧化还原状态是否如此仍存在争议。在这方面，迄今为止已经报道了一种使用烟酰胺腺嘌呤二核苷酸（磷酸）（NAD（P）H）和黄素腺嘌呤二核苷酸（FAD）的寿命特征来计算荧光寿命诱导的氧化还原比（FLIRR）的创新算法。我们扩展了 FLIRR 方法并提出了新的结果，其中包括各种酶（如 NAD（P）H、FAD 和黄素单核苷酸（FMN））的 FLIM 数据。我们的算法对 NAD（P）H 自发荧光使用双指数拟合程序，对黄素信号使用三指数拟合。通过扩展 FLIRR 方法，我们引入了 FLIRR1，其为与蛋白结合的 FAD 相关的蛋白结合的 NAD（P）H，FLIRR2 为与游离（未结合）FAD 相关的蛋白结合的 NAD（P）H，以及 FLIRR3 为与蛋白结合的 FMN 相关的蛋白结合的 NAD（P）H。我们将扩展的 FLIRR 与代谢指数（定义为蛋白结合的 NAD（P）H 与游离 NAD（P）H 的比值）进行了比较。发现对于肿瘤细胞和正常细胞，FLIRR1 的统计学差异最大。
