Shtengel Gleb, Galbraith James A, Galbraith Catherine G, Lippincott-Schwartz Jennifer, Gillette Jennifer M, Manley Suliana, Sougrat Rachid, Waterman Clare M, Kanchanawong Pakorn, Davidson Michael W, Fetter Richard D, Hess Harald F
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA.
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3125-30. doi: 10.1073/pnas.0813131106. Epub 2009 Feb 6.
Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.
要了解细胞的分子尺度结构，需要确定特定蛋白质的三维位置，其精度要与其纳米级长度尺度相匹配。现有的电子显微镜和光学显微镜技术在分子特异性或分辨率方面都存在局限性。在此，我们介绍干涉光激活定位显微镜（iPALM），它是光激活定位显微镜与单光子、同步多相干涉测量法的结合，可提供亚20纳米的三维蛋白质定位，并具有最佳的分子特异性。我们展示了对25纳米微管直径的测量，分辨出背侧和腹侧质膜，并可视化内质网和黏附复合物中整合素受体的排列，这种三维蛋白质组织以前仅通过电子显微镜得以解析。因此，iPALM填补了电子断层扫描和光学显微镜之间的空白，实现了细胞纳米结构的分子特异性和分辨率。
