Key Laboratory of Molecular Nanostructure and Nanotechnology, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2020 Sep 1;92(17):12088-12096. doi: 10.1021/acs.analchem.0c02821. Epub 2020 Aug 12.
Stimulated emission depletion (STED) nanoscopy provides subdiffraction resolution while preserving the benefits of fluorescence confocal microscopy in live-cell imaging. However, there are several challenges for multicolor STED nanoscopy, including sophisticated microscopy architectures, fast photobleaching, and cross talk of fluorescent probes. Here, we introduce two types of nanoscale fluorescent semiconducting polymer dots (Pdots) with different emission wavelengths: CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]) Pdots and PDFDP (poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}---{2,5-bis (,'-diphenylamino)-1,4-phenylene}]) Pdots, for dual-color STED bioimaging and cellular tracking. Besides bright fluorescence, strong photostability, and easy bioconjugation, these Pdots have large Stokes shifts, which make it possible to share both excitation and depletion beams, thus requiring only a single pair of laser beams for the dual-color STED imaging. Long-term tracking of cellular organelles by the Pdots has been achieved in living cells, and the dynamic interaction of endosomes derived from clathrin-mediated and caveolae-mediated endocytic pathways has been monitored for the first time to propose their interaction models. These results demonstrate the promise of Pdots as excellent probes for live-cell multicolor STED nanoscopy.
受激发射耗散（STED）纳米显微镜在提供亚衍射分辨率的同时，保留了荧光共聚焦显微镜在活细胞成像中的优势。然而，多色 STED 纳米显微镜存在几个挑战，包括复杂的显微镜结构、快速的光漂白和荧光探针的串扰。在这里，我们介绍了两种具有不同发射波长的纳米级荧光半导体聚合物点（Pdots）：CNPPV（聚[2-甲氧基-5-（2-乙基己氧基）-1,4-（1-氰基乙烯基-1,4-亚苯基）]）Pdots 和 PDFDP（聚[{9,9-二己基-2,7-双（1-氰基乙烯基）芴}-{2,5-双（,'-二苯基氨基）-1,4-亚苯基}]）Pdots，用于双色 STED 生物成像和细胞跟踪。除了明亮的荧光、强的光稳定性和易于生物偶联外，这些 Pdots 还具有较大的斯托克斯位移，这使得可以共享激发和耗尽光束，因此仅需要一对激光束即可进行双色 STED 成像。已经在活细胞中实现了 Pdots 对细胞细胞器的长期跟踪，并首次监测了由网格蛋白介导和小窝蛋白介导的内吞途径衍生的内体的动态相互作用，以提出它们的相互作用模型。这些结果表明 Pdots 作为活细胞多色 STED 纳米显微镜的优异探针具有广阔的应用前景。
