Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling.
Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling.
复制标题
使用单抗体标记延时成像的单分子定位显微镜。
DOI：
10.1002/cpz1.908
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hu,YingS
中科院分区：
文献类型：
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作者：
Perera,Thilini;Gunasekara,Hirushi;Hu,YingS
在单分子定位显微镜（SMLM）中，免疫荧光（IF）染色会影响重建超分辨率图像的质量。然而，优化IF染色仍然具有挑战性，因为IF染色是一个一步不可逆的过程。通过可逆结合的样品标记提供了一种替代策略，但此类技术需要显著的技术进步来增强标记的解离而不牺牲其结合特异性。在这篇文章中，我们介绍了单抗体标记的时间推移成像。我们的多功能技术利用市售染料偶联抗体。该方法控制抗体浓度，以捕获亚细胞靶标的单抗体标记，从而通过标记过程实现SMLM。我们进一步证明了双色单抗体标记可以提高样品标记密度。新方法允许在单抗体水平和细胞环境内评估抗体结合。这份全面的指南提供了单抗体标记实验的延时成像的分步说明，并使单抗体标记技术能够应用于广泛的靶点。版权所有© 2023作者。当前方案由Wiley Periodicals LLC出版。基本方案1：单抗体标记的样品制备基本方案2：单分子定位显微镜的数据采集替代方案：使用OptoSplit II方程的双色单抗体标记基本方案3：图像分析
In single‐molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one‐step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time‐lapse imaging of single‐antibody labeling. Our versatile technique utilizes commercially available dye‐conjugated antibodies. The method controls the antibody concentrations to capture single‐antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual‐color single‐antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single‐antibody level and within the cellular environment. This comprehensive guide offers step‐by‐step instructions for time‐lapse imaging of single‐antibody labeling experiments and enables the application of the single‐antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sample preparation for single‐antibody labelingBasic Protocol 2: Data acquisition for single‐molecule localization microscopyAlternate Protocol: Dual‐color single‐antibody labeling using OptoSplit II equationBasic Protocol 3: Image analysis