Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.单分子超分辨率显微镜（single-molecule super-resolution microscopy）能够以远低于衍射极限（diffraction limit）的精度，对活细胞内经荧光标记的蛋白质进行成像。本研究通过利用通常被舍弃的拟合信息，滤除来自虚拟光片（virtual-'light-sheet'）——即以显微镜焦平面为中心的薄体积区域——上方或下方的荧光基团（fluorophores），实现了单分子超分辨率显微镜的三维切片成像。我们开发了一款易用程序，以开源ImageJ插件（open-source ImageJ plug-in）的形式实现，可快速对校准样本进行分析以定义并使用此类虚拟光片。此外，该插件几乎可在所有现有二维超分辨率成像设备上流畅运行。通过对衍射极限光斑（diffraction-limited spots）应用经过充分表征的宽度与振幅阈值（可用于调节虚拟光片的厚度），即可实现超分辨率图像的光学切片。这可使成像质量在定性与定量层面均得到提升：通过滤除离焦荧光基团，超分辨率图像的对比度得以提升并可显现局部特征；仅保留焦平面附近的荧光基团后，虚拟光片单分子定位显微镜（virtual-'light-sheet' single-molecule localisation microscopy）可提升所有发射荧光基团被检测、拟合与定量评估的概率。