Spatial Calcium Imaging of Intestinal Interstitial Cells

Colonic muscles were pinned with the CM layer facing upward to the bottom of a 60 mm dish coated with Sylgard (Dow Corning, Midland, MI) and perfused with KRB solution (37°C) for 1 hr before experiments were begun. Ca²⁺ imaging was performed on ICC-IM in situ with an Eclipse E600FN microscope (Nikon Inc., Melville, NY, USA) equipped with a 40-60x 1.0 CFI Fluor lens (Nikon instruments INC, NY, USA). GCaMP6f was excited at 488 nm (T.I.L.L. Polychrome IV, Grafelfing, Germany). Using this acquisition configuration, the pixel size was 0.225 μm. Image sequences of Ca²⁺ transients in ICC-IM were collected at 33 fps with TILLvisION software (T.I.L.L. Photonics GmbH, Grafelfing, Germany). Movement artefacts were stabilized digitally with custom made Volumetry software (10 , 51 (link)–53 (link)) prior to analysis of Ca²⁺ transients. For experiments involving pharmacological treatments, control image sequences were collected for 20-30 sec, and then KRB solution containing the drug concentration to be tested was perfused into the bath for 12-15 mins before another 20-30 sec period of imaging was performed.