Extended Data Figure 6: Deflection of primary cilia in the presence of 50 nM [Ca²⁺] reveals focal-plane-dependent artefact present in ‘top view’ imaging conditions; saturation controls for sensor.

a–c, An mIMCD cell cilium was repeatedly deflected in a low (50 nM) [Ca²⁺] solution. The same flow stimulus (same pipette) was applied to the cilium while imaging in different focal planes. Top panels, experimental arrangement and fluorescence images of the cilium; red dashed lines indicate the focal plane for each set of images. Middle panels (a′–c′), average fluorescence intensity change for F_GECO1.2 (green) and F_mCherry (red) during deflection (black). Lower panels (a′′–c′′), ratio change (blue) during deflection (black). a, Primary cilium near its attachment to the cell. b, At a slightly higher focal plane, deflection enlarges the cross section of the cilium. c, A focal plane ~1 μm above the cell surface. Different segments of the same cilium were imaged upon deflection. Note that the artefact increases with larger cross section changes. Thus, top view imaging of cilia is fraught with two interrelated artefacts: (1) at high z-resolution (0.8 μm), the section of the cilia being imaged changes upon deflection, thus conflating fluorescence changes from different regions of the cilium; (2) at lower z-resolution, the path length, fluorescent indicator volume, and optical properties of the cilium above and below the image plane all change upon deflection and thus contribute to the apparent [Ca²⁺] reporter changes. d, e, Digitonin permeabilization indicates that the Arl13b–mCherry–GECO1.2 sensor is not saturated in measurements of primary cilia and kinocilia. Scale bars, 2 μm. d, Top: mIMCD cell cilium deflected by fluid flow containing 10 μM digitonin. Bottom left: F_mCherry (red) decreased owing to cilia motion (black). F_GECO1.2 (green) rose ~1 s later, as permeabilization initiated Ca²⁺ influx. Right: F_GECO1.2/F_mCherry increased ~4.2-fold upon permeabilization. Representative of 13 videos × 400 frames. e, Kinocilia of E15 cochlear hair cells. Digitonin (10 μM) increased the kinocilium’s normalized F_GECO1.2/F_mCherry ratio by 4.6-fold. Representative of 6 videos × 400 frames. Similar results were obtained in P3 hair cell kinocilia (data not shown).