Lehmann Martin, Lichtner Gregor, Klenz Haider, Schmoranzer Jan
Leibniz Institut für Molekulare Pharmakologie (FMP) & Freie Universität Berlin, Robert-Roessle-Straße 10, 13125, Berlin, Germany.
J Biophotonics. 2016 Jan;9(1-2):161-70. doi: 10.1002/jbio.201500119. Epub 2015 May 13.
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.
精确的基于多色单分子定位的显微镜技术（SMLM）需要具有兼容光化学和光谱特性的明亮探针，以便在纳米尺度上分辨不同的分子种类。多色SMLM的准确性还受到颜色通道串扰和色差对准误差的挑战。这些限制限制了已知的可逆切换有机染料在优化多色SMLM中的适用性。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。我们在不同光谱区域中鉴定出八种新型染料，可实现高质量的dSTORM成像。其中，光谱相近的染料CF647和CF680构成了一对最佳染料，用于基于光谱解混的、无配准的多色dSTORM，且串扰较低。将这对染料与单独激发的CF568相结合，我们进行了三色dSTORM成像，以观察内吞机制和细胞骨架成分的相对纳米级分布。
