美国Eikon Therapeutics公司的Daniel J. Anderson课题组在研究中取得进展。他们提出了斜线扫描照明能够在溶液和活细胞中进行广泛、准确和敏感的单蛋白测量。相关论文发表在2025年2月18日出版的《自然—方法学》杂志上。

在这里，该团队解决了斜线扫描（OLS）的这些局限性，这是一种基于单物镜的机器人照明和检测方式，可以在大视场内实现纳米级空间分辨率和亚毫秒级时间分辨率。课题组研究人员证明，OLS可以捕获活细胞中高达14 μm²s^-1的蛋白质运动。该团队进一步扩展了OLS与溶液中SMT的效用，用于单分子测量配体-蛋白质相互作用和蛋白质-蛋白质相互作用的破坏，以纯化蛋白质为主题。

该课题组人员通过展示光漂白后的双色SMT， STORM和单分子荧光恢复来说明OLS的多功能性。OLS为基础研究、药物筛选和系统生物学研究所需的快速、高通量、单分子蛋白质功能研究铺平了道路。

研究人员表示，细胞生物学研究的一个理想工具将使活细胞内分子活动的无损测量成为可能。单分子定位显微镜（SMLM）技术，如单分子跟踪（SMT），能够在细胞中进行原位测量，但历史上受到时空分辨率和吞吐量之间必要权衡的限制。

附：英文原文

Title: Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells

Author: Driouchi, Amine, Bretan, Mason, Davis, Brynmor J., Heckert, Alec, Seeger, Markus, Silva, Mait Bradley, Forrest, William S. R., Hsiung, Jessica, Tan, Jiongyi, Yang, Hongli, McSwiggen, David T., Song, Linda, Sule, Askhay, Abaie, Behnam, Chen, Hanzhe, Chhun, Bryant, Conroy, Brianna, Elliott, Liam A., Gonzalez, Eric, Ilkov, Fedor, Isaacs, Joshua, Labaria, George, Lagana, Michelle, Larsen, DeLaine D., Margolin, Brian, Nguyen, Mai K., Park, Eugene, Rine, Jeremy, Tang, Yangzhong, Vana, Martin, Wilkey, Andrew, Zhang, Zhengjian, Basham, Stephen, Ho, Jaclyn J., Johnson, Stephanie, Klammer, Aaron A., Lin, Kevin, Darzacq, Xavier, Betzig, Eric, Berman, Russell T., Anderson, Daniel J.

Issue&Volume: 2025-02-18

Abstract: An ideal tool for the study of cellular biology would enable the measure of molecular activity nondestructively within living cells. Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells but have historically been limited by a necessary tradeoff between spatiotemporal resolution and throughput. Here we address these limitations using oblique line scan (OLS), a robust single-objective light-sheet-based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a large field of view. We show that OLS can be used to capture protein motion up to 14μm²s^-1 in living cells. We further extend the utility of OLS with in-solution SMT for single-molecule measurement of ligand–protein interactions and disruption of protein–protein interactions using purified proteins. We illustrate the versatility of OLS by showcasing two-color SMT, STORM and single-molecule fluorescence recovery after photobleaching. OLS paves the way for robust, high-throughput, single-molecule investigations of protein function required for basic research, drug screening and systems biology studies.

DOI: 10.1038/s41592-025-02594-6

Source: https://www.nature.com/articles/s41592-025-02594-6