Figure 3. Membrane potential sensitivity, photostability and phototoxicity of Ap3-SHG assessed in cortical neurons.

(a) Membrane potential sensitivity of SHG signals from FM4-64 and Ap3 assessed by voltage clamp combined with point scan in cortical neurons. Shown are SHG signal changes at each voltage step from a holding potential of −65 mV as means±s.e.m. (black, n=28 points from 4 cells for FM4-64 and 38 points from 8 cells for Ap3), together with linear fit curves (red). (b) SHG signal changes on action potential in neurons. SHG signals were monitored by a point-scan protocol at the soma of patch-clamped neurons in brain slices (scale bars, 2% and 10 ms for FM4-64 and 1% and 10 ms for Ap3) with simultaneous electrophysiological recording (scale bars, 20 mV and 10 ms). Data are shown as means (black)±s.e. m. (light grey) (n=5 cells for FM4-64 and 6 cells for Ap3). (c) Photostability of Ap3-SHG signals in neurons. The SHG signal photostability index was calculated by comparing the mean SHG signal intensities at the initial 5 ms and those 30 ms later during 40-ms-long continuous laser illumination. Individual data points (circles) are shown with boxes corresponding to the 25th, 50th and 75th percentile (n=38 cells for Ap3 an 28 cells for FM4-64). (d) Reduced phototoxicity of Ap3 SHG imaging in neurons. Neurons in brain slices were loaded with Ap3 (500 μM) or FM4-64 (200 μM) and frame-scan imagings was performed with a long pixel dwell time (100 μs per pixel) to induce photodamage while monitoring membrane potential changes. Individual data points (circles) are shown with boxes corresponding to the 25th, 50th and 75th percentile (n=8 for both Ap3 and FM4-64).