Sperm temporally sample chemoattractant molecules. (A and B) Identical paired stimuli of 12.5 pM resact were delivered by photolysis of 100 nM caged resact; stimulus functions (top), V_m signals (middle), and Ca²⁺ signals (bottom) are shown. Flashes in A: single 50% (blue), single 100% (black), and paired 50% with Δt = 50 ms (red). Flashes in B: single 50% (blue), single 100% (black), and paired 50% with Δt = 100 ms (red) or 200 ms (green). (C) Decrease of sampling efficacy of V_m (red) and Ca²⁺ (black) signals with increasing Δt. Three experiments as shown in A and B were analyzed. The V_m and Ca²⁺ signals evoked by a full intensity flash were normalized to 0 (baseline) and 1 (peak amplitude). The signal amplitudes evoked by paired half-intensity flashes were scaled to these two values. (D) Sampling efficacy depends on stimulus strength. The low stimulus strength regimen was achieved using 10 nM caged resact and flashes of 25 and 50% (black; n = 4). The high stimulus strength regimen was achieved using 100 nM caged resact and flashes of 50 and 100% (red; n = 3). (E) Sperm temporally sample changes in cGMP concentration. Ca²⁺ signals were evoked by photolysis of caged cGMP. Flashes: single 10% (blue), single 20% (black), and two 10% with a Δt of 30 ms (red), 75 ms (green), or 150 ms (magenta). (F) V_m signals evoked by paired stimuli of cGMP. Flashes: single 8% (blue), single 16% (black), and two 8% with Δt = 50 ms (red). (Inset) V_m signals of identical amplitude evoked by cGMP (green) and resact (orange). (G) Sperm count single resact molecules. Ca²⁺ signals were evoked by releasing ∼625 fM resact (blue), single full intensity flash (1.25 pM; black), and two half-intensity flashes (625 fM each) with Δt = 30 ms (red) or 75 ms (green). Error bars show SDs. Arrows indicate time of flashes.