Fig. 7. Fluorescence recordings of photo-evoked action potentials in hippocampal organotypic slices co-expressing JEDI-2P-Kv and ChroME-ST, using 2P-TF-CGH.

a Schematic overview of the experimental protocol. JEDI-2P-Kv expressing organotypic hippocampal slices were prepared as described (for more details, refer to Fig. 3 and “Methods” section). At p18, slices were transduced with AAV9.CaMKII.ChroME-ST.P2A.H2B.BFP. Experiments were performed between p21 and p28 using a 940 nm, 250 kHz repetition rate laser source. b Cross-sections of hippocampal organotypic slices co-expressing the genetically encoded voltage indicator JEDI-2P-Kv and the soma-targeted channelrhodopsin ChroME-ST in the dentate gyrus (n = 4 slices from 2 independent slice cultures). Channelrhodopsin-expressing cells were identified according to their nuclear-localised fluorescence (see “Methods” section). Scale bar represents 50 μm. c Simultaneous optical and electrophysiological recordings demonstrating that APs can be evoked and imaged using a single excitation spot (12 µm diameter, power density 0.02 mW µm⁻² (2.5 mW per cell), 15 ms strobed illumination at 5 Hz). The red bar represents the illumination epoch. Asterisks indicate detected APs. d Zoom on simultaneous optical and electrophysiological recordings of one AP. e Probability of evoking and recording APs as a function of power density. AP probability is calculated as the number of APs evoked and detected at each power (power density: 0.01–0.09 mW μm⁻²). Data is plotted as mean ± SEM of recordings obtained for 33 repetitions. Probabilities greater than 100 % indicate that more than one action potential was detected in a given trial. f Simultaneous optogenetic activation of ChroME-ST and voltage imaging of JEDI-2P-Kv. 10 cells were targeted simultaneously using 2P-TF-CGH and imaged at 500 Hz. Here we show an example trace from one cell when the 10 cells were targeted sequentially (upper panel) or simultaneously (lower panel). In both the sequential and multi-cell acquisitions, APs were only evoked/recorded in the cells co-expressing JEDI-2P-Kv and ChroME-ST. All data was acquired using laser C (940 nm, 250 kHz repetition rate) and camera A. Source data are provided as a Source Data file.