Digital straightening allows reconstruction of IFT in beating flagella even when IFT particles are not directly visible. (A) Still frame from a 100-Hz dual-color, high-frame-rate video micrograph of a cell expressing IFT52::eGFP (IFT marker) and SMP1::mCh (flagellar membrane marker). Individual IFT particles are not visible in the flagellum and flagellar motion is significantly blurred at this exposure time (10 ms). (B) SMP1::mCh image used for flagellum tracing (red) and the flagellum midline (white line) determined by thresholding. (C) Digitally straightened view of the flagellum, showing the same frame as in A straightened using the midline in B. (D) Tangent angle at different distances along the flagellum, represented as a graph and a color-coded bar, showing the same frame as in A. (E) Kymograph of flagellum tangent angle over time for a 50-frame section of the dual-color, high-frame-rate video micrograph. (F) Power spectrum over time, calculated from the flagellum tangent angle kymograph, for the full-length video micrograph. (G) Kymograph of IFT52::eGFP and SMP1::mCh fluorescent signal over time for the full-length video micrograph. Animated version of A and C–G in Video 3.