使用Cer-SiR对高尔基体动力学进行受激发射损耗成像：一种用于活细胞的双组分、光稳定、高密度脂质探针。
STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.
作者信息
Erdmann Roman S, Toomre Derek, Schepartz Alanna
机构信息
Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520, USA.
Department of Chemistry, Yale University, New Haven, CT, 06520, USA.
出版信息
Methods Mol Biol. 2017;1663:65-78. doi: 10.1007/978-1-4939-7265-4_6.
Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.
对活细胞进行长时间延时超分辨率成像需要一种标记策略，该策略将明亮、光稳定的荧光团与高密度定位探针相结合。脂质是理想的高密度定位探针，因为它们的丰度比大多数膜结合蛋白高100倍以上，同时还能界定细胞器的边界。在这里，我们描述了Cer-SiR，一种双组分、高密度脂质探针，其光稳定性极佳。Cer-SiR通过两种组分的生物正交反应在细胞中生成：一种用反式环辛烯标记的神经酰胺脂质（Cer-TCO）和一种具有反应性、光稳定的硅罗丹明染料（SiR-Tz）。这些组分在活细胞的高尔基体中组装形成Cer-SiR。Cer-SiR对细胞功能无害，以高密度定位于高尔基体，并且具有足够的光稳定性，能够通过三维共聚焦或长时间延时受激发射损耗显微镜观察高尔基体的结构和动态。
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