Shroff Hari, White Helen, Betzig Eric
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
Curr Protoc Cell Biol. 2008 Dec;Chapter 4:Unit 4.21. doi: 10.1002/0471143030.cb0421s41.
Key to understanding a protein's biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (approximately 200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA-FP) tags necessary to achieve PALM images at approximately 20 nm resolution in 5 to 30 min in fixed cells.
理解蛋白质生物学功能的关键在于准确确定其在细胞内的空间分布。尽管荧光蛋白标记物能够以分子精度靶向特定蛋白质，但当使用传统的衍射极限光学器件对所得融合蛋白进行成像时，许多此类信息会丢失。作为回应，出现了几种能够实现低于衍射极限（约200纳米）分辨率的成像方式。在此，描述了使用光活化定位显微镜（PALM）对生物结构进行单通道和双通道超分辨率成像。所讨论的示例聚焦于黏附复合体：在细胞与其基质之间的界面处形成的密集、充满蛋白质的组装体。特别强调了在固定细胞中以约20纳米分辨率在5至30分钟内获得PALM图像所需的仪器设备和光活化荧光蛋白（PA-FP）标签。
