An aldehyde- crosslinking mitochondrial probe for STED imaging in fixed cells

• 成果类型: Article
• 署名作者: Chen, Jingting; Stephan, Till; Gaedke, Felix; Liu, Tianyan; Li, Yiyan; Schauss, Astrid; Chen, Peng; Wulff, Veronika; Jakobs, Stefan; Juengst, Christian; Chen, Zhixing
• 署名单位: Peking University; University of Gottingen; UNIVERSITY GOTTINGEN HOSPITAL; University of Cologne; Peking University; University of Gottingen
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9118
• DOI: 10.1073/pnas.2317703121
• 发表日期: 2024-05-07
• 关键词: electron-microscopy; fluorescence; light; Fixation; ultrastructure; organelles

摘要

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in superresolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable superresolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane - potential - dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria - specific probe, PK Mito Orange FX (PKMO FX), which features a fixation - driven cross - linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative superresolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.