Aix Marseille Université, CNRS, INP UMR7051, NeuroCyto, 13005 Marseille, France; Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France.
Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France; Université Paris Saclay, CNRS, Institut des Sciences Moléculaires d'Orsay, 91405 Orsay, France.
Cell Rep Methods. 2023 Sep 25;3(9):100571. doi: 10.1016/j.crmeth.2023.100571. Epub 2023 Sep 1.
Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.
单分子定位显微镜（SMLM）可以使用随机光学重建显微镜（STORM）或 DNA 点积累用于纳米级形貌成像（PAINT）等技术达到亚 50nm 的分辨率。在这里，我们通过将发射荧光分为两个相机来实现更快的多色 SMLM 的两种方法：同时双色 DNA-PAINT（S2C-DNA-PAINT），在每个相机上分别对光谱分离的红色和远红色成像链进行成像；和光谱解混 dSTORM（SD-dSTORM），在两个相机上出现光谱接近的远红色荧光团，然后通过解混来识别。使用 S2C-DNA-PAINT 作为低串扰的参考，我们使用三种类型的样品评估 SD-dSTORM 串扰：不同大小的 DNA 折纸纳米棒、单个标记靶细胞或标记多个靶细胞的细胞。然后，我们评估串扰是否会影响对生物相关亚衍射图案的检测。将这些方法扩展到三维采集和 SD-dSTORM 进行三色成像，我们表明光谱解混是一种用于稳健和多功能多色 SMLM 研究的有吸引力的选择。
