近日，美国西北大学Vadim Backman团队开发了染色质中的三色单分子定位显微镜。相关论文于2025年3月17日发表在《光：科学与应用》杂志上。

超分辨率显微镜彻底改变了人们观察传统光学显微镜衍射极限以下结构的能力，尤其适用于研究染色质等复杂的生物靶标。染色质表现出一种分层组织，具有不同长度尺度（从纳米到微米）的结构隔间和域。单分子定位显微镜（SMLM）方法，如STORM，对于在核小体上水平研究染色质至关重要，因为它们能够靶向决定染色质组织的表观遗传标记。染色质的多标记成像对于揭示其结构复杂性是必要的。然而，这些努力受到高密度核环境的挑战，这会影响抗体结合亲和力、扩散性和非特异性相互作用。优化缓冲条件、荧光团稳定性和抗体特异性对于实现有效的抗体偶联物至关重要。

研究组展示了一种顺序免疫标记方案，能够在密集的核环境中可靠地进行三色研究。该协议将多路复用的定位数据集与稳健的分析算法相结合，利用来自一个目标的定位作为距离、密度和多标签联合亲和力测量的种子点，以探索所有三个目标的复杂组织。应用这种多路复用算法来分析距离和关节密度表明，异染色质和常染色质不是不同的区域，但转录和常染色质的定位与异色簇的外围相结合。这项工作是致密核环境分子成像的关键一步，因为多标记能力能够以更高的精度研究复杂的多组分系统，如染色质。

附：英文原文

Title: Three-color single-molecule localization microscopy in chromatin

Author: Acosta, Nicolas, Gong, Ruyi, Su, Yuanzhe, Frederick, Jane, Medina, Karla I., Li, Wing Shun, Mohammadian, Kiana, Almassalha, Luay, Wang, Geng, Backman, Vadim

Issue&Volume: 2025-03-17

Abstract: Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

DOI: 10.1038/s41377-025-01786-1

Source: https://www.nature.com/articles/s41377-025-01786-1