德国欧洲分子生物学实验室（EMBL）的Jonas Ries研究组，发现核孔可作为定量超分辨显微镜的通用参考标准。2019年10月刊的《自然—方法学》发表了这一成果。

研究人员利用蛋白质在核孔复合物中的定型排列作为原位参考结构来表征各种显微镜模式的性能。研究人员创建了四个基因组编辑的细胞系，其中使用mEGFP、SNAP标签、HaloTag或光可转换荧光蛋白mMaple内源标记了核孔蛋白Nup96。研究人员证明了它们在以下三个方面的使用：（1）作为用于校准和质量控制的三维分辨率标准，（2）量化绝对标记效率，以及（3）作为分子计数的精确参考标准。这些细胞系将使更广泛的研究团体能够评估其显微镜和标签的质量，并实现定量的绝对测量。

据了解，定量荧光和超分辨率显微镜通常受到数据质量不足或人为因素的限制。在这种情况下，至关重要的是要有生物学上相关的对照样品来确定基准并优化显微镜、标签和成像条件的质量。

附：英文原文

Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Author: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cie?liński, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya Kasuba, Sarah Janice Hoerner, Yiming Li, Yu-Le Wu, Markus Mund, Ulf Matti, Pedro Matos Pereira, Ricardo Henriques, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jan Ellenberg & Jonas Ries 

Issue&Volume: Volume 16 Issue 10

Abstract:

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

DOI: 10.1038/s41592-019-0574-9

Source: https://www.nature.com/articles/s41592-019-0574-9