2.11. HILO microscopy

HEK293T cells were seeded onto 24 mm coverslips (Cat #: 631–1584, VWR, Radnor, PA, USA) and transfected with 500 ng of GLP-1R-GFP or GIPR-GFP over 24 h. HILO image sequences were acquired with a custom-built TIRF microscope (Cairn Research) based on an Eclipse Ti2 (Nikon, Tokyo, Japan) equipped with an EMCCD camera (iXon Ultra, Andor), a 488 nm diode laser, a hardware Perfect Focus System, a TIRF iLas2 module, and a 100× oil-immersion objective (NA 1.49, Nikon). Coverslips were mounted onto metal imaging chambers with a plastic seal and filled with imaging medium (HBSS supplemented with 10 mM of HEPES). The objective and samples were maintained at 37 °C in a heated enclosure. Images were acquired on MetaMorph software (Molecular Devices) using a frame exposure of 50–200 ms with an image acquired before ligand stimulation and a subsequent image taken every 30 s thereafter, up to 20 min. All of the images were analyzed using ImageJ.