基于荧光的成像方法，使用mt-Keima进行体外和体内线粒体测量。,Nature Protocols

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基于荧光的成像方法，使用mt-Keima进行体外和体内线粒体测量。
Nature Protocols ( IF 13.1 ) Pub Date : 2017-Aug-01 , DOI: 10.1038/nprot.2017.060
Nuo Sun , Daniela Malide , Jie Liu , Ilsa I Rovira , Christian A Combs , Toren Finkel

线粒体吞噬是一种细胞过程，可选择性去除受损，陈旧或功能异常的线粒体。线粒体缺陷被认为是导致正常衰老以及各种神经退行性疾病和心血管疾病的原因。用于体内检测线粒体吞噬的先前方法麻烦，不灵敏且难以量化。我们创建了一个表达pH依赖性荧光蛋白mt-Keima的转基因小鼠模型，以便更容易地评估线粒体。凯玛（Keima）是一种pH敏感，双重激发比例荧光蛋白，对溶酶体蛋白酶也具有抗性。在线粒体的生理pH（pH 8.0）下，较短波长的激发占主导。线粒体化后的酸性溶酶体（pH 4.5）中，mt-Keima逐渐过渡到更长波长的激发。在这个协议中我们描述了如何在18小时的时间内监视活细胞中的线粒体通量，以及如何使用mt-Keima探针定量线粒体。该协议还描述了如何使用共聚焦显微镜观察从mt-Keima转基因小鼠获得的活组织中的线粒体。使用此协议，可以在组织收集后的前60分钟内可靠地对mt-Keima探针成像。我们还描述了如何在受激发射损耗（STED）显微镜下应用mt-Keima技术，该技术可以潜在地提供更高分辨率的图像。通常，活细胞对mt-Keima进行延时荧光成像的大概时间是20小时。对于来自mt-Keima小鼠的组织的共聚焦分析，整个过程通常不超过60分钟，而STED成像通常需要2小时。

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A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.

Mitophagy is a cellular process that selectively removes damaged, old or dysfunctional mitochondria. Defective mitophagy is thought to contribute to normal aging and to various neurodegenerative and cardiovascular diseases. Previous methods used to detect mitophagy in vivo were cumbersome, insensitive and difficult to quantify. We created a transgenic mouse model that expresses the pH-dependent fluorescent protein mt-Keima in order to more readily assess mitophagy. Keima is a pH-sensitive, dual-excitation ratiometric fluorescent protein that also exhibits resistance to lysosomal proteases. At the physiological pH of the mitochondria (pH 8.0), the shorter-wavelength excitation predominates. Within the acidic lysosome (pH 4.5) after mitophagy, mt-Keima undergoes a gradual shift to longer-wavelength excitation. In this protocol, we describe how to monitor mitophagic flux in living cells over an 18-h time frame, as well as how to quantify mitophagy using the mt-Keima probe. This protocol also describes how to use confocal microscopy to visualize mitophagy in living tissues obtained from mt-Keima transgenic mice. With this protocol, the mt-Keima probe can reliably be imaged within the first 60 min after tissue collection. We also describe how to apply mt-Keima with stimulated emission depletion (STED) microscopy, which can potentially provide substantially higher-resolution images. Typically, the approximate time frame for time-lapse fluorescence imaging of mt-Keima is 20 h for living cells. For confocal analysis of tissue from an mt-Keima mouse, the whole procedure generally takes no longer than 60 min, and the STED imaging usually takes <2 h.

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