Fig. 3.

(a-c) High-speed (1016 Hz frame rate) two-photon imaging of a cultured neuron expressing SF-Venus-iGluSnFR.A184V. a) RuBi-glutamate was uncaged for 10 msec. at each of two 5-micron spots (arrowheads) on two adjacent dendrites (Supp. Video 2). Color saturation denotes the glutamate transient amplitude; grey tones indicate no evoked response. Color hue denotes response timing (see scale bars). The yellow line indicates the axis of the kymograph shown in (c). Representative example of 4 trials. b) Recorded traces at the centers of the uncaging locations shown in (a). c) Top: Kymograph showing the response amplitude over time along the dendrite surrounding uncaging location 1. Traces are approximate maximum-likelihood solutions¹⁸ for a spatially multiplexed recording acquired with a high-power 1030 nm fiber laser. These recordings show a single trial without averaging. Bottom: simulation (Methods) of diffusion of glutamate at the surface of a planar coverslip surrounded by 3D solution, following uncaging in a 5 μm spot with a 0.8 NA beam at time 0. Intensity scale is log-transformed. (d-f) Recording of visually-evoked spine transients in isolated SF-Venus-iGluSnFR.A184S labeled dendrites in mouse visual cortex, imaged at 1030 nm excitation. See also Supp. Video 3. d) Motion-aligned average image. e) Mean responses (top) and tuning curves (bottom) of ROIs indicated in d, for 20 trials of each of the 8 oriented moving grating stimuli. Dark lines/markers denote mean, shaded regions/error bars denote SEM. Black bar denotes the stimulus period. f) pixel intensity of ROI 1 for eight consecutive stimulus presentations of different directions, illustrating detectable responses in single trials. Colored bars denote stimuli as in (e). The spine response is selective to the stimulus orientation. (d-f) representative example of 7 mice.