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Abstract：
Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution 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 super-resolved 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 super-resolution 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.
摘要：
化学固定标本的荧光标记,尤其是免疫标记,在超分辨率成像中起着至关重要的作用，因为它提供了一种方便的方法来可视化细胞结构，如线粒体或具有高细节的生物分子的分布。尽管开发了各种不同的探针，可以对活细胞中的线粒体进行超分辨受激发射消耗（STED）成像，这些膜电位依赖性荧光团中的大多数在化学固定后不能很好地保留在线粒体中。缺乏合适的线粒体探针限制了线粒体对活细胞样品的STED成像。在这项研究中,我们引入了一种线粒体特异性探针，PKMitoOrangeFX（PKMOFX），它具有固定驱动的交联基序，并积聚在线粒体内膜中。它在化学固定和775nm的有效耗尽后表现出高荧光保留，能够在醛固定之前和之后进行纳米成像。我们证明了该探针与常规免疫标记和通常用于固定样品的荧光标记的其他策略的相容性。此外，我们证明了PKMOFX促进了相关的超分辨率光学和电子显微镜，通过特征性的线粒体模式实现多色荧光图像和透射EM图像的相关性。我们的探针进一步扩展了用于纳米分辨率的多模态显微镜的线粒体工具包。
