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Automated ExEm-spFRET Microscope

Published online by Cambridge University Press:  21 February 2022

Han Sun
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Chenshuang Zhang
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Ye Yuan
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Lu Gao
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Shixian Zhai
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Hongce Chen
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Qilin Tang
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China
Zhengfei Zhuang*
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China SCNU Qingyuan Institutes of Science and Technology Innovation Co., Ltd., Qingyuan511517, China
Tongsheng Chen*
Affiliation:
Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China SCNU Qingyuan Institutes of Science and Technology Innovation Co., Ltd., Qingyuan511517, China
*
*Corresponding author: Zhengfei Zhuang, E-mail: zhuangzf@scnu.edu.cn; Tongsheng Chen, E-mail: chentsh@scnu.edu.cn; chentsh126@126.com
*Corresponding author: Zhengfei Zhuang, E-mail: zhuangzf@scnu.edu.cn; Tongsheng Chen, E-mail: chentsh@scnu.edu.cn; chentsh126@126.com

Abstract

Excitation–emission-spectral unmixing-based fluorescence resonance energy transfer (ExEm-spFRET) microscopy exhibits excellent robustness in living cells. We here develop an automatic ExEm-spFRET microscope with 3.04 s of time resolution for a quantitative FRET imaging. The user-friendly interface software has been designed to operate in two modes: administrator and user. Automatic background recognition, subtraction, and cell segmentation were integrated into the software, which enables FRET calibration or measurement in a one-click operation manner. In administrator mode, both correction factors and spectral fingerprints are only calibrated periodically for a stable system. In user mode, quantitative ExEm-spFRET imaging is directly implemented for FRET samples. We implemented quantitative ExEm-spFRET imaging for living cells expressing different tandem constructs (C80Y, C40Y, C10Y, and C4Y, respectively) and obtained consistent results for at least 3 months, demonstrating the stability of our microscope. Next, we investigated Bcl-xL-Bad interaction by using ExEm-spFRET imaging and FRET two-hybrid assay and found that the Bcl-xL-Bad complexes exist mainly in Bad-Bcl-xL trimers in healthy cells and Bad-Bcl-xL2 trimers in apoptotic cells. We also performed time-lapse FRET imaging on our system for living cells expressing Yellow Cameleon 3.6 (YC3.6) to monitor ionomycin-induced rapid extracellular Ca2+ influx with a time interval of 5 s for total 250 s.

Type
Software and Instrumentation
Copyright
Copyright © The Author(s), 2022. Published by Cambridge University Press on behalf of the Microscopy Society of America

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