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使用荧光探针进行逐步标记：MINFLUX成像和追踪的通用方法。

Gradual labeling with fluorogenic probes: A general method for MINFLUX imaging and tracking.

作者信息

Yao Longfang, Si Dongjuan, Chen Liwen, Li Shu, Guan Jiaxin, Zhang Qiming, Wang Jing, Ma Jiong, Wang Lu, Gu Min

机构信息

School of Artificial Intelligence Science and Technology, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China.

Institute of Photonic Chips, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China.

出版信息

Sci Adv. 2025 May 23;11(21):eadv5971. doi: 10.1126/sciadv.adv5971. Epub 2025 May 21.

DOI:10.1126/sciadv.adv5971
PMID:40397744
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12094232/
Abstract

Minimal photon fluxes (MINFLUX) nanoscopy excels in nanoscale protein studies but lacks a universal method for simultaneous imaging and live-cell tracking in dense cellular environments. Here, we developed a general strategy, gradual labeling with fluorogenic probes for MINFLUX (GLF-MINFLUX) imaging and tracking. In GLF-MINFLUX, membrane-permeable small-molecule fluorogenic dye with protein-induced "off/on" switching is gradually labeled, located, and bleached, enabling sequential positioning and tracking of individual proteins. GLF-MINFLUX reveals continuous microtubules with 2.6-nanometer localization precision, offering substantially improved precision (1.7-fold), acquisition (2.2-fold), and target density (3-fold) compared to conventional MINFLUX with Alexa Fluor 647. GLF-MINFLUX also enabled the three-dimensional localization of translocase of the outer mitochondrial membrane 20 proteins within mitochondrial clusters and dual-channel nanoscale imaging of endogenous neuronal microtubules and microfilaments. GLF-MINFLUX allowed live-cell single-protein tracking with 7.8-nanometer precision at ~200-microsecond temporal resolution, revealing distinct diffusion behaviors and rates between the basal membrane and filopodia. GLF-MINFLUX, requiring only tuning of probe concentration, offers molecular-level insights into protein functions.

摘要

最小光子通量（MINFLUX）纳米显微镜在纳米级蛋白质研究方面表现出色，但在密集细胞环境中缺乏同时成像和活细胞追踪的通用方法。在此，我们开发了一种通用策略，即使用用于MINFLUX成像和追踪的荧光探针进行逐步标记（GLF-MINFLUX）。在GLF-MINFLUX中，具有蛋白质诱导的“关/开”切换的膜渗透性小分子荧光染料被逐步标记、定位和漂白，从而能够对单个蛋白质进行顺序定位和追踪。GLF-MINFLUX以2.6纳米的定位精度揭示了连续的微管，与使用Alexa Fluor 647的传统MINFLUX相比，其精度显著提高（1.7倍）、采集速度提高（2.2倍）、目标密度提高（3倍）。GLF-MINFLUX还实现了线粒体外膜转位酶20蛋白在线粒体簇内的三维定位以及内源性神经元微管和微丝的双通道纳米级成像。GLF-MINFLUX能够以约200微秒的时间分辨率进行7.8纳米精度的活细胞单蛋白追踪，揭示了基底膜和丝状伪足之间不同的扩散行为和速率。GLF-MINFLUX仅需调整探针浓度，即可提供对蛋白质功能的分子水平见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/fe78a9ccfcaa/sciadv.adv5971-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/0d211b68b59f/sciadv.adv5971-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/d9cc58bb9a24/sciadv.adv5971-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/3f328df73a2c/sciadv.adv5971-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/9d302466cc93/sciadv.adv5971-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/e2823fa745c2/sciadv.adv5971-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/fe78a9ccfcaa/sciadv.adv5971-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/0d211b68b59f/sciadv.adv5971-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/d9cc58bb9a24/sciadv.adv5971-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/3f328df73a2c/sciadv.adv5971-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/9d302466cc93/sciadv.adv5971-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/e2823fa745c2/sciadv.adv5971-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/10d3/12094232/fe78a9ccfcaa/sciadv.adv5971-f6.jpg

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本文引用的文献

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Direct optical measurement of intramolecular distances with angstrom precision.直接以埃精度测量分子内距离。
Science. 2024 Oct 11;386(6718):180-187. doi: 10.1126/science.adj7368. Epub 2024 Oct 10.
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Photoactivatable Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy.光活化的碳和硅罗丹明及其在 MINFLUX 纳米显微镜中的应用。
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Ångström-resolution fluorescence microscopy.埃（Ångström）分辨率荧光显微镜。
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Direct observation of motor protein stepping in living cells using MINFLUX.利用 MINFLUX 在活细胞中直接观察马达蛋白的运动。
Science. 2023 Mar 10;379(6636):1010-1015. doi: 10.1126/science.ade2676. Epub 2023 Mar 9.
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MINFLUX dissects the unimpeded walking of kinesin-1.MINFLUX剖析驱动蛋白-1的自由行走。
Science. 2023 Mar 10;379(6636):1004-1010. doi: 10.1126/science.ade2650. Epub 2023 Mar 9.
6
Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores.通过自发快速闪烁荧光团实现的加速MINFLUX纳米显微镜技术。
Small. 2023 Mar;19(12):e2206026. doi: 10.1002/smll.202206026. Epub 2023 Jan 15.
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Choosing the Probe for Single-Molecule Fluorescence Microscopy.选择单分子荧光显微镜的探针。
Int J Mol Sci. 2022 Nov 29;23(23):14949. doi: 10.3390/ijms232314949.
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Super-resolution imaging and quantitative analysis of microtubule arrays in model neurons show that epothilone D increases the density but decreases the length and straightness of microtubules in axon-like processes.超分辨率成像和对模型神经元中微管阵列的定量分析表明，埃坡霉素 D 增加了轴突样突起中微管的密度，但降低了其长度和直线度。
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DNA-PAINT MINFLUX nanoscopy.DNA-PAINT 微光纳米显微镜技术。
Nat Methods. 2022 Sep;19(9):1072-1075. doi: 10.1038/s41592-022-01577-1. Epub 2022 Sep 1.
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A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy.用于活细胞纳米显微镜的无笼组光活化荧光团的通用设计。
Nat Chem. 2022 Sep;14(9):1013-1020. doi: 10.1038/s41557-022-00995-0. Epub 2022 Jul 21.