FLIM-FRET measurement of OSM-3. (A) Representative cilia images of osm-3 null mutant and animals expressing FLIM-FRET constructs. For osm-3 null animals, cilia are visualized with mScarlet-tagged endogenous DYF-11. Arrows indicate the ciliary base. Arrowheads indicate the junctions between the middle and distal segments. Scale bar, 5 μm. Dyf, dye-filling defective; N ≥ 100. (B) Examples of fluorescence intensity decay curve showing different FRET efficiency at different regions of amphid cilia. 10 representative worms of mScarlet::osm-3::gfp strain were measured. (C) Frequency distribution of GFP fluorescence lifetimes measured from images as shown in Fig. 1 D. 10 representative worms of each strain were measured. The plots were fit with a Gaussian distribution. (D) Statistics of GFP fluorescence lifetimes measured from different regions of amphid neurons. The GFP fluorescence lifetimes of other regions are normalized to soma of each strain. More than 14 worms were measured for each strain. Comparisons were performed between OSM-3::GFP::mScarlet and mScarlet::OSM-3::GFP. n.s., not significant; ***P < 0.001 by unpaired Student’s t tests. Data are mean ± SD.