Shtengel Gleb, Wang Yilin, Zhang Zhen, Goh Wah Ing, Hess Harald F, Kanchanawong Pakorn
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
Mechanobiology Institute, National University of Singapore, Singapore.
Methods Cell Biol. 2014;123:273-94. doi: 10.1016/B978-0-12-420138-5.00015-X.
Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250 nm laterally and >500 nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20 nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750 nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.
通过荧光显微镜可以高灵敏度和特异性地观察细胞中的许多生物分子。然而，传统光学显微镜的分辨率受衍射限制，横向约为200 - 250纳米，轴向大于500纳米。在此，我们描述了基于光开关荧光团单分子定位分析的超分辨率方法（PALM：光激活定位显微镜）以及我们最近的三维（3D）方法（iPALM：干涉PALM），该方法能够在所有三个维度上以优于20纳米的分辨率进行成像。讨论了其实施的注意事项、在多色成像中的应用以及将iPALM成像深度扩展至约750纳米的最新进展。由于超分辨率荧光显微镜的空间分辨率与电子显微镜（EM）的分辨率趋于一致，使用这两种方法对同一标本进行直接成像变得可行。这对于实验的交叉验证可能特别有用，因此，我们还描述了最近为相关超分辨率荧光和EM开发的方法。
