Optosplit image splitter

TIRFM used an Olympus IX81 inverted microscope equipped with an oil immersion 100× PlanApo objective with a numerical aperture of 1.45. The microscope was equipped with a LG-TIRFM (TIRF Labs, Cary, NC) system coupled to an Argon-Helium laser with three lines; 488, 543, and 633 nm as previously described^{68 (link)}. The 488 nm line was used to excite GFP (NMDA receptors) and the 543 to excite Cav1-mCherry. We introduced an Optosplit image splitter (Cairn Research, Kent, UK) before the camera (Ixon Andor) to collect simultaneous images from GFP (525 nm) and mCherry (580 nm).

CHO cells were plated at 1.8 × 10⁵cells/dish on 1% BSA-coated 35 mm glass-bottom dishes (Matek) and grown for 24 h before transfection with HA-HER2-WT and HA-HER3-WT full-length plasmids using Viafect (Promega), according to the manufacturer's protocol. Forty-eight hours post-transfection, cells were serum-starved for 1 h, then treated for 1 h with 30 μM AC3573 compound or 0.3% DMSO in serum-free medium. Cells were then labelled with 0.5 nM HER2-Alexa488 Affibody and 15 nM HER3-CF640R Affibody or 14 nM NRG-CF640R for 7 min at 37°C and washed with serum-free medium before prompt imaging. Single-molecule images were acquired using an Axiovert 200M microscope with TIRF illuminator (Zeiss, U.K.), with a 100× oil-immersion objective (α-Plan-Fluar, NA = 1.45; Zeiss, U.K.) and an EMCCD (iXon X3; Andor, U.K.). The 488 nm and 642 nm lines of a LightHub laser combiner (Omicron Laserage GmbH) were used to illuminate the sample and an Optosplit Image Splitter (Cairn Research) was used to separate the image into its spectral components as described previously [48 (link)]. The field of view of each channel for single-molecule imaging was 80 × 30 µm. All single-molecule time series data were analysed using the multidimensional analysis software described previously [49 (link)]. Calculation of colocalisation and τON were performed as previously described [50 (link)].