Biophysical Analytics, Deutsches Rheuma-Forschungszentrum (DRFZ), 10117 Berlin, Germany.
Dynamic and Functional in vivo Imaging, Freie Universität Berlin, 14163 Berlin, Germany.
Int J Mol Sci. 2019 Nov 7;20(22):5565. doi: 10.3390/ijms20225565.
In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.
在过去的几年中，免疫系统的细胞代谢经历了复兴，因为人们已经清楚地认识到，它不仅负责细胞的能量供应，还影响许多信号通路，从而影响多种细胞功能。普遍存在的辅酶 NADH 和 NADPH（NAD(P)H-FLIM）的无标记荧光寿命成像使得能够在活细胞和组织中监测细胞代谢，并且已经应用于研究生理和病理条件下的代谢变化。然而，由于 NAD(P)H 依赖性酶在细胞中的复杂分布，其分布随时间不断变化，因此，彻底解释 NAD(P)H-FLIM 结果，特别是解析各种酶对整体代谢活性的贡献，仍然具有挑战性。我们开发了一个基于角度相似性的系统框架，用于分析细胞和组织的 NAD(P)H-FLIM 数据，该框架基于细胞中高度丰富的 NAD(P)H 依赖性酶的普遍有效的参考系统。通过使用我们的分析框架，我们不仅可以获取有关整体代谢活性的信息，即游离 NAD(P)H 与酶结合 NAD(P)H 的分数，还可以以亚细胞分辨率在特定时间点识别样本中主要活跃的酶。我们通过将 NAD(P)H-FLIM 应用于基质样细胞系来验证该方法的性能，并鉴定出与细胞质相比，在细胞核中活跃的不同酶类。由于无标记 NAD(P)H-FLIM 的基于相位向量的系统分析框架既可以在体外又可以在体内应用，因此它保留了独特的能力，可以在真实环境中以亚细胞分辨率进行基于酶的动态代谢研究。
