Supplementary Figure 3: Calibration of the synOptopatch constructs.

a, Simultaneous fluorescence and manual patch-clamp recordings of PSPs and action potentials evoked by presynaptic optogenetic stimulation. Blue, 10 ms blue light stimulation; red, whole-cell single-trial unfiltered fluorescence; black, patch-clamp recordings. b, Overlay of mean optically and electrically recorded PSP waveforms from a single cell (n = 9 repeats). c, Quantification of fluorescence versus postsynaptic potential for synOptopatch recordings (n = 10 neurons, R² = 0.9). d, Quantification of photocurrent of QuasAr2 under red and blue illumination (red: 400 W cm^–2; blue: 10 ms, 20 – 120 mW cm^–2). e,f, Quantification of optical crosstalk of blue illumination into QuasAr2 fluorescence. e, Neurons expressing QuasAr2 were exposed to continuous excitation at 640 nm and pulses of illumination at 488 nm (10 ms, 60 mW cm^–2) (n = 17 neurons). f, Quantification of crosstalk amplitude as a function of blue light intensity. The shading represents the range of blue light intensity used for optogenetic stimulation of primary neurons. Error bars represent s.e.m. g, SynOptopatch fluorescence recordings of EPSPs were stable for at least 2 h. h, Photobleaching of QuasAr2 in the synOptopatch assay. A neuron was illuminated for 30 minutes continuously at 640 nm, 400 W cm^–2 and probed at 60 s intervals with blue light to induce PSPs (2 pulses of 10 ms, 2 Hz, 60 mW cm^–2). i, Stability of PSP waveform recorded as a function of duration of continuous red illumination. (In h,i, n = 3 neurons; representative data are shown.) All shaded error bars, s.e.m.