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 μ m 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.

中文翻译：

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空间滤镜及其在三维单分子定位显微镜中的应用

单分子定位显微镜（SMLM）可以对细胞结构进行成像，其分辨率比光学显微镜的衍射极限低五到十倍。它最初是作为二维技术引入的，该技术基于投影到xy成像平面上的单个发射器的定位。荧光发射器的轴向位置的确定只能通过附加信息来实现。在这里，我们报告了一种方法（空间过滤器SMLM（SFSMLM）），该方法允许通过使用两个空间过滤器来确定纳米级荧光分子和纳米颗粒的轴向位置，这两个空间过滤器放置在两个其他相同的发射检测通道中。SFSMLM允许轴向定位范围为ca。1.5μm，轴向定位精度为15-30 nm。