Single-molecule imaging was performed 24–48 h after RNA injection using total internal reflection fluorescence (TIRF) microscopy. Manually devitellinized oocytes were placed on high-refractive-index coverglass (n = 1.78) and imaged through a 100× 1.65-NA oil immersion objective (Olympus). 13 × 13–µm frames showing low density (50–250 individual puncta per frame) to minimize the risk of false colocalization were imaged. EGFP-tagged VSDs and full-length subunits were excited with a PhoxX 488 (60 mW) laser, and mCherry-tagged PDs and full-length subunits were excited with a 593-nm diode-pumped solid-state (DPSS) laser. z488/594-rpc polychroic (Chroma) was used as the excitation filter; 525/50 and 629/53 emission filters were used for EGFP and mCherry, respectively. mCherry and EGFP were excited sequentially. Videos of 800 frames (∼200 for mCherry and 600 for EGFP) were captured at the rate of 20 Hz with the iXon DU-887 EMCCD camera (Andor Technology).
The bleaching videos of the PD tagged with EGFP were acquired in a similar way, using Nikon 100× 1.49 NA oil immersion TIRF objective. Devitellinized oocytes were placed on 35-mm µ-dishes with glass bottom (n = 1.52; IBIDI). They were excited with a 488-nm laser. The light path contained a 525/50-nm emission filter. The videos were captured with iXon DU-897 EMCCD camera (Andor Technology) at 10 Hz.
The bleaching videos of the PD tagged with EGFP were acquired in a similar way, using Nikon 100× 1.49 NA oil immersion TIRF objective. Devitellinized oocytes were placed on 35-mm µ-dishes with glass bottom (n = 1.52; IBIDI). They were excited with a 488-nm laser. The light path contained a 525/50-nm emission filter. The videos were captured with iXon DU-897 EMCCD camera (Andor Technology) at 10 Hz.