Multichannel wide-field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra

作者：Du, M.; Mai, Z.; Yang, F.; Lin, F.; Wei, L.; Chen, T.^*

来源：Journal of Microscopy, 2018, 269(1): 66-77.

DOI：10.1111/jmi.12609

• excitation-emission spectra
• multichannel wide-field microscope
• quantitative FRET imaging
• spectral crosstalks
• system correction factor
• spectral unmixing

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter-based multichannel wide-field microscope for ExEm unmixing-based FRET imaging (m-ExEm-spFRET) containing a constant system correction factor (f(sc)) for a stable system. We performed m-ExEm-spFRET with four- and two-wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (R-C). We also performed m-ExEm-spFRET imaging for single living cells coexpressing CFP-Bax and YFP-Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin-treated cells when R-C were larger than 1, indicating that staurosporin induced significant oligomerisation. @@@ Lay description We recently developed a spectral wide-field microscope by integrating a liquid crystal tunable filter (LCTF) into a wide-field microscope for ExEm unmixing-based quantitative FRET measurement (ExEm-spFRET). However, the very low transmittance of LCTF made this system inapplicable to living cells with low expression levels of FPs, and the measured FRET efficiency by using ExEm-spFRET method on this system were slightly larger than those measured by other's methods. We here introduce a system correction factor to modify the ExEm-spFRET method (m-ExEm-spFRET) and set up a filter-based multichannel wide-field microscope for quantitative m-ExEm-spFRET measurement. Moreover, implementation of m-ExEm-spFRET method with as few as two-wavelength excitation on our system for living cells expressing FRET constructs exhibits stable and accurate results, which is very important for fast quantitative m-ExEm-spFRET imaging in living cells.

• 出版日期2018-1
• 单位华南师范大学