Workflow for quantitative IFT analysis in beating flagella. (A) Still frame from a 200 Hz dual-color, high-frame-rate video micrograph of a cell expressing 3×mNG::IFT172 (IFT marker) and SMP1::mCh (flagellar membrane marker). (B) Still frame from a 200 Hz dual-color, high-frame-rate video micrograph of a cell expressing 3×mNG::IFT172 (IFT marker) in fluorescence and phase contrast. (C) SMP1::mCh image used for flagellum tracing (red) and the flagellum midline (white line) determined by thresholding. (D) Digitally straightened view of the flagellum showing the same frame as in A straightened using the midline in B. (E) Tangent angle at different distances along the flagellum, represented as a graph and a color-coded bar, showing the same frame as in A. (F) Kymograph of flagellum tangent angle over time for a 100-frame section of the dual-color, high-frame-rate video micrograph. (G) Power spectrum over time, calculated from the flagellum tangent angle kymograph, for the full-length video micrograph. (H) Kymograph of 3×mNG::IFT172 and SMP1::mCh fluorescent signal over time for the full-length video micrograph. IFT trains are readily visible. Animated version of A and D–H in Video 2.