Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Molecular Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
Methods Mol Biol. 2023;2643:65-84. doi: 10.1007/978-1-0716-3048-8_5.
Peroxisomes are crucial organelles that occur in almost all eukaryotes. Well known are their roles in various metabolic processes, such as hydrogen peroxide detoxification and lipid metabolism. Recent studies indicated that peroxisomes also have several non-metabolic functions, for instance, in stress response, signaling, and cellular ageing. In mammalian cells, the small size of peroxisomes (~200 nm, near the diffraction limit) hinders unveiling peroxisomal structures by conventional light microscopy. However, in the yeast Hansenula polymorpha, they can reach up to 1.5 μm in diameter, depending on the carbon source. To study the localization of peroxisomal proteins in cells in more detail, super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy can be used. STED enables fast (live-cell) imaging well beyond the diffraction limit of light (30-40 nm in cells), without further data processing. Here, we present optimized protocols for the fluorescent labeling of specific peroxisomal proteins in fixed and living cells. Moreover, detailed measurement protocols for successful STED imaging of human and yeast peroxisomes (using antibodies or genetic tags labeled with dyes) are described, extended with suggestions for individual optimizations.
过氧化物酶体是几乎所有真核生物中都存在的重要细胞器。众所周知，它们在各种代谢过程中发挥着重要作用，如过氧化氢解毒和脂质代谢。最近的研究表明，过氧化物酶体还具有几种非代谢功能，例如在应激反应、信号转导和细胞衰老中。在哺乳动物细胞中，过氧化物酶体的体积较小（~200nm，接近衍射极限），这使得传统的光学显微镜难以揭示过氧化物酶体的结构。然而，在酵母汉逊酵母中，过氧化物酶体的直径可以根据碳源的不同达到 1.5μm。为了更详细地研究细胞中过氧化物酶体蛋白的定位，可使用超分辨率成像技术，如受激发射损耗（STED）显微镜。STED 可以在无需进一步数据处理的情况下，超越光的衍射极限（细胞中为 30-40nm），实现快速（活细胞）成像。在这里，我们提供了优化的固定和活细胞中特定过氧化物酶体蛋白荧光标记的方案。此外，还描述了成功进行人类和酵母过氧化物酶体的 STED 成像（使用抗体或用染料标记的遗传标签）的详细测量方案，并提供了针对各个优化的建议。
