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《工程(英文)》 >> 2022年 第16卷 第9期 doi: 10.1016/j.eng.2021.12.010

基于谐波的多光子扫描结构光照明超分辨显微成像

a College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen 518060, China
b Department of Laser Medicine, First Medical Center of PLA General Hospital, Beijing 100853, China
c Department of Bioengineering and COMSET, Clemson University, Clemson, SC, 29634, United States

收稿日期: 2021-02-06 修回日期: 2021-06-10 录用日期: 2021-12-22 发布日期: 2022-02-16

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摘要

本文提出了一种利用多光子非线性效应的多光子结构光照明超分辨显微成像(multiphoton-structured illumination microscopy, mP-SIM)技术,可实现比线性SIM 更高的成像分辨率。通过扫描正弦结构的照明光激发样本产生多光子荧光信号或谐波信号,从而利用由光学非线性效应产生的非正弦结构信号光的谐波来提高分辨率。本文提出了mP-SIM 理论以重建系统的超分辨图像。如果考虑多光子非线性效应的所有高阶谐波(阶数足够高,即m足够大),则mP-SIM 在理论上可实现无限分辨率。通过实验证实了双光子荧光(2P)-SIM 和二次谐波产生(second harmonic generation, SHG)-SIM 分别可实现86 nm 和72 nm的横向分辨率。对细胞内染色F-肌动蛋白和小鼠尾腱胶原纤维成像,进一步验证了mP-SIM 的超分辨成像能力。该方法不但适用于多光子荧光超分辨显微成像,而且适用于非标记的如SHG等超分辨显微成像;重要的是,该方法不需要特定的荧光团或高功率激发光,可直接用于商用mP显微系统来实现超分辨成像。

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