Raw Microscopy Data for: Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane.

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities – an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This unique combination enables quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieve versatile combination of SMLM and SIM with up to three channels. We deploy this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enables long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties can be correlated with nanoscale cytoskeletal organization and dynamics.

全内反射荧光（Total internal reflection fluorescence, TIRF）显微镜凭借极高的空间与时间分辨率，为解析细胞质膜中蛋白质的功能组织提供了强有力的研究手段。然而传统全内反射荧光照明的强度分布为高斯强度分布（Gaussian intensity profile），且通常会因叠加的干涉条纹而出现劣化，这会阻碍强度的精确定量——而精确定量是单分子定位显微镜（single-molecule localization microscopy, SMLM）定量分析的关键前提。本研究将采用标准πShaper实现的平场照明，与搭载适配快速超分辨率结构照明显微镜（fast super-resolution structured illumination microscopy, SIM）的空间光调制器（spatial light modulator）所实现的多角度全内反射照明相结合。这种独特的组合方案可实现照明高度均匀的定量多色单分子定位显微镜成像。通过搭载优化分像光学元件的双相机系统，我们可实现最多三通道的单分子定位显微镜与结构照明显微镜的灵活联用。我们将该系统应用于基于单分子强度分析与单分子弗斯特共振能量转移（single-molecule Förster resonance energy transfer, smFRET）的受体化学计量比的稳健检测。此外，均匀照明还支持细胞表面受体的长期追踪与定位显微镜（long-term tracking and localization microscopy, TALM）成像，能够揭示细胞质膜上受体迁移率与可及性的空间异质性。通过结合长期追踪与定位显微镜与结构照明显微镜，可将空间与分子层面的异质扩散特性与纳米尺度细胞骨架的组织及动态变化相关联。