Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells

作者：Jung Seung Ryoung^*; Deng Yi; Kushmerick Christopher; A**ury Charles L; HiIle Bertil^*; Koh Duk Su

来源：Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(25): E5706-E5715.

DOI：10.1073/pnas.1717724115

• Single-molecule live-cell
• imaging
• metabolite
• ATP
• oxygen scavenger
• fluorescence microscopy

The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by similar to 20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and a-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH(2) and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.

• 出版日期2018-6-19