Illustration on the spectral wide-field microscope. 405: ET405/20x bandpass excitation filter (405 nm excitation); 436: BP436/20 bandpass excitation filter (436 nm excitation); D455: E455Ipv2 dichroic mirror (455 nm dichroic mirror, D455); 470: a cube contains a BP470/40 bandpass excitation filter (470 nm excitation) and a FT495 dichroic mirror (495 nm dichroic mirror, D495); 510: a cube contains a BP510/17 bandpass excitation filter (510 nm excitation) and a DFT520 dichroic mirror (520 nm dichroic mirror, D520); DD: a cube contains filters for donor excitation and donor emission; AA: a cube contains filters for acceptor excitation and acceptor emission; DA: a cube contains filters for donor excitation and acceptor emission; LCTF: liquid crystal tunable filter (Varispec LCTF, VIS-10-20-STD, CRI, Cambridge); CCD1: CCD camera for quantitative EES-FRET measurement; CCD2: CCD camera for three-cube-based quantitative FRET measurement; a: for CCD1 detection; b: for CCD2 detection.