Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University.
Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University; Becker & Hickl GmbH.
J Vis Exp. 2021 Jul 31(173). doi: 10.3791/62708.
Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.
黏度是生物膜的一个重要物理性质，因为它是调节活细胞形态和生理状态的关键参数之一。众所周知，肿瘤细胞的质膜在组成、结构和功能特性上都有显著改变。除了葡萄糖和脂质代谢失调外，这些特定的膜特性还帮助肿瘤细胞适应恶劣的微环境，并对药物治疗产生耐药性。在这里，我们展示了使用荧光寿命成像显微镜（FLIM）在活的癌细胞培养物中顺序成像细胞代谢和质膜黏度的方法。通过检测内源性代谢辅助因子（如还原型烟酰胺腺嘌呤二核苷酸 NAD(P)H 和氧化黄素）的荧光来进行代谢评估。黏度是使用荧光分子转子（一种合成的对黏度敏感的染料）来测量的，其荧光寿命强烈依赖于其周围环境的黏度。这两种技术相结合，使我们能够更好地理解肿瘤细胞膜状态和代谢特征之间的联系，并可视化化疗引起的变化。
