Figure 2. Optically assessing cupula deflection–the mechanical input of lateral line hair cells. A, Side projection of a neuromast expressing GFP in hair cells with the surface of the cupula stained with AlexaFluro-350-WGA. Notice how the kinocilia extend approximately half way up the cupula. The staining was not evenly distributed across the surface of the cupula. Scale bar, 10 μm. B, Schematic of the model used to calculate angular deflections of the cupula from a pivot at its base. The translational deflection for a given stimulus pressure was measured at several distances z above the apical surface of the hair cell. C, Three representative frames of the stained cupula at z = 15 μm (B, blue dashed line) at rest and deflected by a positive (pos) and negative (neg) pressure step. The angular deflection was calculated as tan−1 (Δx/z), where Δx was the translation in the center of mass of the staining from the rest position (dashed red line indicated center position). D, Cupula motion in space over the course of the entire experiment can be extracted from these data. Stimulation along the x-axis (parallel to the fish), led to increasingly strong translational displacements toward the tip of the cupula along the x-axis and hardly induced motion along the orthogonal, y-axis. Scale bars: x, y, 10 μm; z, 5 μm. E, Traces showing x displacement as a function of time at four different z distances and for a variety of pressure steps (bottom trace). The image sequences were obtained at 20 Hz. F, The x displacements to positive and negative pressure steps of increasing magnitude (black, green, and red, corresponding to stimuli delivered in boxes shown in E). Note that x displacement is directly proportional to z at any pressure, indicating that the proximal part of the cupula indeed acts as a beam, deflecting at a pivot point at its base. Several measurements within this rigid region of the cupula could therefore be averaged. G, Example of a calibration curve relating the stimulus pressure to the angular deflection (average of several Δx measurements in the “rigid” part of the cupula), gray shading indicated SEM, see Materials and Methods. These relations were generally linear. H, Translational displacement (same as in F) of the cupula at z = 10 μm for an intermediate (left) and strong (right) deflection step. Although the intermediate pressure step led to a deflection within 1 sample point (50 ms), an additional slow small creep was apparent in some of the stronger steps. I, Two examples of translational displacements of cupulae at z = 10 μm to saturating pressure steps in which the applied pressure led to a steady deflection within 1 sample point (50 ms, indicated by markers).