Tirf illuminator

Single-molecule images were acquired using an Axiovert 200M microscope with a TIRF illuminator (Zeiss, UK) and incorporating a 100 x oil-immersion objective (α-Plan-Fluar, NA = 1.45; Zeiss, UK) and an EMCCD (iXon X3; Andor, UK). Samples were illuminated with a 638 nm laser (100 mW, Vortran) fed into the microscope via a polarisation maintaining triple laser combiner (Oz Optics) . Alternatively, the 640 nm lines of a Vortran Combiner or of an Andor Revolution Laser Combiner were used. A wrap-around incubator (Pecon XL S1) was used to maintain a constant temperature of 37°C. The field of view of each channel was 80 × 30 μm. Data were acquired at 20 Hz for 30 s. Images were saved in HDF5 format for subsequent processing using custom-designed software. All Single-Molecule time series data were analysed using the multidimensional analysis software described previously (Rolfe et al., 2011 (link)).

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)].