超越衍射屏障彻底改变了现代荧光显微镜。然而,统计抽样的内在局限性,可同时分析的通道的数量,硬件要求,和样品制备程序仍然是其在应用生物医学研究中广泛传播的障碍。这里,我们提出了一种基于自动多模态显微镜和超分辨率技术的新管道,采用易于获得的材料和仪器,并使用我们实验室开发的开源图像分析软件完成。结果表明,单分子定位显微镜(SMLM)对生物分子相互作用和大分子复合物定位研究的潜在影响。作为示范应用,我们探索了p53-53BP1相互作用的基础,显示了两种蛋白质和原位基础转录机制之间推定的大分子复合物的形成,因此提供了53BP1在维持p53反式激活功能中的直接作用的视觉证据。此外,高含量的SMLM提供了在细胞骨架和线粒体空间中存在53BP1复合物的证据,因此表明存在新的替代53BP1功能来支持p53活性。
Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules\' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-
53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of
53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a
53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative
53BP1 functions to support p53 activity.