Application of stochastic optical reconstruction microscopy in the observation of exosomes
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摘要:
目的 探讨随机光学重构显微镜（STORM）在外泌体观察中的应用价值，并介绍外泌体在随机光学重构显微镜下的成像原理及方法。 方法 实验中在肾性甲旁亢患者原代培养的甲状旁腺细胞的培养液上清中加入EXOQUICK-TC外泌体沉淀剂来获取细胞培养上清中的外泌体，将外泌体膜表面的特异性跨膜蛋白CD63作免疫荧光标记后利用随机光学重构显微镜对外泌体行超高分辨率成像并测量外泌体直径。 结果 实验中随机光学重构显微镜成功的对继发性甲旁亢甲状旁腺细胞原代培养液上清中的外泌体行单分子定位、直径测量及超高分辨率成像。 结论 因随机光学重构显微镜具有独特的光学特性并突破了光学衍射极限的限制，较传统光学显微镜，能获得20~50 nm的分辨率，可对外泌体行单分子精确定位、直径测量、超高分辨率成像。基于STORM的成像优势，相信STORM及其他超高分辨率成像技术将在外泌体及外泌体参与的生物学过程的研究中发挥重要作用。 Abstract:Objective To investigate the application value of stochastic optical reconstruction microscopy (STORM) in the observation of exosomes. Methods In the experiment, exosomes were extracted by adding EXOQUICK-TC to the culture media supernatants of primarily cultured parathyroid cells from patients with hyperparathyroidism. After the immunofluorescence labeling of the specific transmembrane protein CD63 on the surface of exosomes, STORM was applied to perform ultra-high resolution imaging and diameter measurement of exosomes. Results In the experiment, STORM successfully performed the single molecule positioning, diameter measurement and ultra-high resolution imaging of exosomes extracted from the culture media supernatants of parathyroid cells of patients with hyperparathyroidism. Conclusion Compared to conventional optical microscopy, STORM, with its unique optical characteristics which has broken through the optical diffraction limit, has a resolution of 20-50 nm and can perform single-molecular acute positioning, diameter measurement and ultra-high resolution imaging of exosomes. In view of these outstanding features, we believe that STORM and other ultra-high resolution imaging technologies will play vital roles in the subsequent research on exosomes and exosome-related physiological procedures. -
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