Movie S2.

Axial-plane specific photorescue of mGlu₅ receptors by 2PE but not 1PE in HEK cells. Video of GCaMP6s fluorescence in cultured HEK cells co-transfected with mGlu5 receptors. Photocontrol of cell Ca²⁺ occurs regardless of the axial distance for 1PE of alloswitch, and is axial-plane selective for 2PE. The field of view corresponds to image and cell Ca²⁺ trace in Fig. 4B–C in main text. Ca²⁺ events are recorded in different conditions indicated by labels (‘agonist’ = 3 μM quisqualate; ‘+ alloswitch/no light’ = 1 μM alloswitch supplemented in bath in addition to agonist; ‘1PE’ = 405 nm laser diode, 2 μW; ‘2PE’ = NIR pulsed laser, 780 nm, 20 mW; ‘light OFF’ = end of photostimulation) (SI Appendix, Materials and Methods 5.2). Axial position of photostimulation (z in main figures) is indicated between asterisks, and is 30 or 10 μm above cells, or corresponds to the cell imaging plane (0 μm). GCaMP6s fluorescence is excited by raster scanning with a 488 nm Argon laser (100 nW, 343 ms/frame). Frame rate is 0.2 Hz during imaging alone, 0.17 Hz during 1P photostimulation, and 0.12 Hz during 2P photostimulation. Picture size is 129×129 μm.