Fig. 4: Ca²⁺ optopatch for all-optical electrophysiology in cultured neurons and in zebrafish embryonic heart in vivo.

A Average fluorescence trace of cultured rat hippocampal neurons expressing FR-GECO1a or 1c via transient transfection, under constant 561 nm illumination (13 mW/mm²) and 100 ms flash of 470 nm LED illumination (23 mW/mm²). Time-average of fluorescence increase induced by 470 nm illumination for each neuron was quantified and shown in red (FR-GECO1a) or blue (FR-GECO1c) dots (n = 6 for both FR-GECO1a and 1c). B HEK293T cells transiently transfected with CheRiff were illuminated by alternating pulses of 488 nm and 594 nm light of increasing intensities (top). Photocurrents were measured under a voltage clamp at −65 mV (middle). Photocurrents at 594 nm were then normalized to the maximum photocurrent induced by excitation with 488 nm (bottom). Typical light intensities used for optopatch experiments are highlighted in orange (n = 3 cells). Average fluorescence traces of cultured rat hippocampal neurons expressing FR-GECO1a (or 1c)-P2A-CheRiff-eGFP-Kv2.1 (AAV transduction) under constant 594 nm illumination (10 mW/mm²) and 470 nm optical stimulation for 0.6 s at 0.07 mW/mm² (C) or 0.35 mW/mm² (D) (n = 45 neurons for FR-GECO1a; n = 37 neurons for FR-GECO1c). Blue light crosstalk during the stimulation epochs (dotted lines) has been subtracted from the traces. Error bands represent S.E.M. E Isochronal map of calcium wave in an embryonic zebrafish linear heart tube (LHT) at 24 h post-fertilization (hpf). The map was calculated from a spike-triggered average (n = 12 beats). Each isochron is 50 ms, propagating from left to right. Cardiac marker Nkx2.5:ZsYellow marked in green. Scale bar 50 μm. F Mean FR-GECO1c ΔF/F of the LHT in (E). Simultaneous pacing and measurement of Ca²⁺ dynamics in the LHT using FR-GECO1a combined with optical stimulation at 1 Hz using CheRiff (G) and CoChR (H). FR-GECOs were imaged using constant 594 nm illumination at 24–290 mW/mm². Channelrhodopsin was excited using 488 nm light at 24 mW/mm² (25 ms pulse for CoChR, 75 ms pulse for CheRiff). All images were acquired at 33 Hz. Source data are provided in a Source Data file.