Blacker Thomas S, Sewell Michael D E, Szabadkai Gyorgy, Duchen Michael R
Research Department of Cell & Developmental Biology, University College London, London, UK.
UCL Consortium for Mitochondrial Research, University College London, London, UK.
Methods Mol Biol. 2019;1928:365-387. doi: 10.1007/978-1-4939-9027-6_19.
Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.
代谢改变是癌症的一个标志，它既是肿瘤发生的结果，也是肿瘤发生的驱动因素。NAD和NADP氧化还原对在大量相关代谢途径中起关键作用。这些分子以还原形式NADH和NADPH存在时具有内在荧光性。由于激光脉冲激发后发射荧光的平均时间（即荧光寿命）对荧光团局部环境的变化极为敏感，因此对NADH和NADPH的荧光寿命进行成像可为活体肿瘤内部代谢变化的无标记监测提供可能。在此，我们描述了将NAD(P)H荧光寿命成像（FLIM）确立为分析活癌细胞和组织代谢的常规方法所需考虑的生物学、光物理和方法学因素。
