(Peer-Reviewed) Multiplexed stimulated emission depletion nanoscopy (mSTED) for 5-color live-cell long-term imaging of organelle interactome
Yuran Huang 黄宇然 ¹, Zhimin Zhang 张智敏 ¹ ², Wenli Tao 陶汶理 ¹, Yunfei Wei 魏云飞 ³, Liang Xu 徐良 ¹, Wenwen Gong 宫文文 ¹, Jiaqiang Zhou 周嘉强 ⁴, Liangcai Cao 曹良才 ⁵, Yong Liu 刘勇 ⁶, Yubing Han 韩于冰 ¹ ³, Cuifang Kuang 匡翠方 ¹ ² ⁷, Xu Liu 刘旭 ¹ ²
¹ State Key Laboratory of Extreme Photonics and Instrumentation, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310027, China
中国 杭州 浙江大学光电科学与工程学院 极端光学技术与仪器全国重点实验室
² Research Center for Intelligent Chips and Devices, Zhejiang Lab, Hangzhou 311121, China
中国 杭州 之江实验室 智能芯片与器件研究中心
³ Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China
中国 武汉 华中科技大学武汉光电国家研究中心
⁴ Department of Endocrinology and Metabolism, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China
中国 杭州 浙江大学医学院附属邵逸夫医院 内分泌代谢科
⁵ Department of Precision Instruments, Tsinghua University, Beijing 100084, China
中国 北京 清华大学精密仪器系
⁶ College of electronics and information engineering, Shanghai University of Electrical Power, Shanghai 200090, China
中国 上海 上海电力大学 电子与信息工程学院
⁷ ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou 311200, China
中国 杭州 浙江大学杭州国际科创中心
Opto-Electronic Advances
, 2024-07-05
Abstract
Stimulated emission depletion microscopy (STED) holds great potential in biological science applications, especially in studying nanoscale subcellular structures. However, multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation. Here, we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.
By separating live-cell fluorescent probes with similar spectral properties using phasor analysis, our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures. The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.
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