Institute of Complex Systems (ICS-4, Cellular Biophysics), Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Institute of Complex Systems (ICS-5, Molecular Biophysics), Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 201203 Shanghai, People's Republic of China.
Methods Appl Fluoresc. 2020 Mar 19;8(2):025008. doi: 10.1088/2050-6120/ab7e0f.
Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.
单分子定位显微镜（SMLM）允许以低于光学显微镜衍射极限五到十倍的分辨率对细胞结构进行成像。它最初是作为一种二维技术被引入的，其基础是对单发射器的定位，即对 x-y 成像平面的投影。荧光发射器的轴向位置只能通过附加信息来确定。在这里，我们报告了一种方法（空间滤波器单分子定位显微镜（SFSMLM）），该方法允许通过使用两个空间滤波器来确定纳米尺度上荧光分子和纳米粒子的轴向位置，这两个空间滤波器放置在两个否则完全相同的发射检测通道中。SFSMLM 允许在大约 1.5 μm 的范围内进行轴向定位，轴向定位精度为 15-30nm。该技术用于对小细胞结构进行定位和三维成像，例如肌动蛋白丝、囊泡和线粒体。
