二氨基联苯胺光氧化法在透射电子显微镜下观察贴壁培养细胞中的荧光纳米颗粒。

Diaminobenzidine Photooxidation to Visualize Fluorescent Nanoparticles in Adhering Cultured Cells at Transmission Electron Microscopy.

机构信息

Department of Neurosciences, Biomedicine and Movement Sciences, Anatomy and Histology Section, University of Verona, Verona, Italy.

出版信息

Methods Mol Biol. 2023;2566:333-343. doi: 10.1007/978-1-0716-2675-7_27.

DOI:10.1007/978-1-0716-2675-7_27

PMID:36152264

Abstract

Visualizing nanoparticles made of organic material (e.g., polysaccharides, proteins, non-osmiophilic lipids) inside cells and tissues at transmission electron microscopy is a difficult task due to the intrinsic weak electron density of these nanoconstructs, which makes them hardly distinguishable in the biological environment. We describe here a simple protocol to apply photooxidation to fluorescently labeled nanoparticles administered to cultured cells in vitro. The conversion of the fluorescent signal into a granular electron-dense reaction product through light irradiation in the presence of diaminobenzidine makes the nanoparticles clearly visible at the ultrastructural level. Our procedure proved to be reliable with various fluorophores and may be applied to any cell type.

摘要

在透射电子显微镜下观察细胞和组织内由有机材料（例如多糖、蛋白质、非亲脂性脂质）制成的纳米颗粒是一项艰巨的任务，因为这些纳米结构的固有电子密度较弱，使得它们在生物环境中难以区分。我们在这里描述了一种简单的方案，可将体外给予培养细胞的荧光标记纳米颗粒进行光氧化。通过在存在二氨基联苯胺的情况下用光照射将荧光信号转化为颗粒状电子致密反应产物，使得纳米颗粒在超微结构水平上清晰可见。我们的程序已被证明与各种荧光团兼容，并且可以应用于任何细胞类型。