Kadam Parnika, McAllister Ryan, Urbach Jeffrey S, Sandberg Kathryn, Mueller Susette C
Department of Biochemistry, Georgetown University Medical Center; Department of Medicine, Georgetown University Medical Center.
Department of Physics, Georgetown University Medical Center.
J Vis Exp. 2017 Mar 27(121):55177. doi: 10.3791/55177.
Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.
活细胞成像用于在转染的人胚肾293（HEK）细胞中，在用血管紧张素II（Ang II）刺激后，同时捕获血管紧张素1a受体（AT1aR）和细胞内区室的延时图像。用含有标记有增强型绿色荧光蛋白（EGFP）的AT1aR的质粒DNA对HEK细胞进行瞬时转染。用红色荧光染料识别溶酶体。在Ang II刺激后，在激光扫描共聚焦显微镜上捕获活细胞图像，并通过软件随时间进行三维（3D，体素）分析。活细胞成像能够研究受体运输，并避免与固定相关的混淆，特别是避免与EGFP标记的膜受体的丢失或人为位移相关的问题。因此，随着单个细胞随时间被追踪，可以对受体的亚细胞定位进行成像和测量。图像必须足够快速地采集以捕获快速的囊泡运动。然而，在更快的成像速度下，收集到的光子数量会减少。为了提高成像速度，在诸如体素大小等成像参数的选择上也必须做出妥协。活细胞成像的重要应用包括研究蛋白质运输、迁移、增殖、细胞周期、凋亡、自噬以及蛋白质-蛋白质相互作用和动力学等等。
