Fig. 1. Schematic diagram of FRET measurement optical path with free site selection (inset: timing diagram of scanning mirror control voltage)

Fig. 2. Fluorescent lifetime measurement process and iterative fitting process. (a) Fluorescence lifetime measurement and iterative fitting; (b) SSE between fitted curve and measured decay curve after 495 iterations (τ_L ranging from 0.05 ns to 5 ns); (c) fitting results of fluorescence decay curve considering the IRF

Fig. 3. FRET measurement principle based on fluorescence lifetime. (a) FRET measurement principle diagram based on donor fluorescence lifetime before and after acceptor bleaching; (b) donor fluorescence emission spectrum curve (green) and acceptor absorption spectrum curve (red); (c) fluorescence decay curve of donor before and after acceptor photobleaching

Fig. 4. Experimental verification of focus free site selection and positioning. (a) Confocal intensity image of fluorescent microspheres with a diameter of 40 nm; (b) fluorescence decay curves obtained at 0.5 s when the focus is located at position 1 (fluorescent microsphere) and position 2 (background region), respectively; (c) Intensity fluctuation curves obtained at the positions from 1 to 4 over 120 s

Fig. 5. Measurement of the fluorescence lifetime of mitochondria and lipid droplet in COS-7 cells using free-addressing FRET. (a) Confocal image of Nile red-stained COS-7 cells; (b)(c) enlarged images of area within the white dashed box in Fig. 5(a), with orange and blue circles representing the lipid droplets and mitochondrial sites to be tested, respectively; (d) fluorescence decay curves of lipid droplets (orange) and mitochondria (blue); (e) statistics of fluorescence lifetime values of 10 independent mitochondria and lipid droplets

Fig. 6. FRET efficiency measurement of donors and acceptor on the mitochondrial outer membrane. (a)(c) Intensity images of initial donor eGFP fluorescent protein and acceptor mCherry fluorescent protein; (b)(d) intensity images of the donor and acceptor fluorescent proteins after acceptor bleaching; (e) fluorescence decay curves of donor fluorescent protein before and after bleaching of acceptor fluorescent protein; (f) statistical distribution of donor fluorescence lifetime before (gray) and after (red) acceptor bleaching and FRET efficiency obtained from six experimental groups