Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells.
With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E ) and acceptor-to-donor concentration ratio (R C ) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral wide-field microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells.
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Lili
L
Zhang
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Guiqi
G
Qin
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Liuying
L
Chai
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Jiang
J
Zhang
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Fangfang
F
Yang
Fujian Normal University, Institute of Laser and Optoelectronics Technology, Key Laboratory of Optoelectronic Science and Technology for Medicine of Ministry of Education, Fuzhou 350007, China.
Hongqin
H
Yang
Fujian Normal University, Institute of Laser and Optoelectronics Technology, Key Laboratory of Optoelectronic Science and Technology for Medicine of Ministry of Education, Fuzhou 350007, China.
Shusen
S
Xie
South China Normal University, College of Life Science, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Guangzhou 510631, China.
Tongsheng
T
Chen
D058948:Cell Tracking / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D056945:Hep G2 Cells; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation; D000069416:Intravital Microscopy / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000648:ultrastructure*