Novel organic dyes for multicolor localization-based super-resolution microscopy
Novel organic dyes for multicolor localization-based super-resolution microscopy
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DOI：
10.1002/jbio.201500119
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发表时间：
2016-01-01
影响因子：
2.8
通讯作者：
Schmoranzer, Jan
中科院分区：
文献类型：
--
作者：
Lehmann, Martin;Lichtner, Gregor;Schmoranzer, Jan
精确的单分子定位显微镜（SMLM）需要具有兼容的光化学和光谱特性的明亮探针，以分辨纳米级的不同分子种类。彩色通道串扰和色度对准误差进一步挑战了彩色SMLM的准确性。这些限制限制了已知可逆切换有机染料对于优化多色SMLM的适用性。在这里，我们测试了28种市售染料对超分辨已知细胞纳米结构的适用性。我们确定了八种不同光谱范围的新型染料，可以实现高质量的dSTORM成像。在这些染料中，光谱接近的染料CF 647和CF 680包括用于具有低串扰的基于光谱解混的无配准的BLDSTORM的最佳染料对。将该染料对与单独激发的CF568组合，我们进行了3色dSTORM以成像内吞机制和细胞骨架的组分的相对纳米级分布。基于单分子定位的超分辨率显微镜（SMLM）的主要限制是合适的光可切换荧光染料的可用性。通过筛选28种市售染料，确定了不同光谱范围的新型染料，这些染料非常适合于具有低串扰的双色和三色SMLM。这些新的染料用于成像亚细胞组分的相对纳米级分布。
Precise multicolor single molecule localization-based microscopy (SMLM) requires bright probes with compatible photo-chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super-resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing-based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3-color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.[GRAPHICS]A major limitation of multicolor single molecule localization based super-resolution microscopy (SMLM) is the availability of suitable photo-switchable fluorescent dyes. By screening 28 commercially available dyes, novel dyes in different spectral regimes were identified that are well suited for dual and triple color SMLM with low crosstalk. These novel dyes are employed to image the relative nanoscale distribution of sub-cellular components.