Cfi fluor lens

Muscle sheets isolated from the colon, stomach, and jejunum (∼5.0 × 10.0 mm) were pinned down and perfused with 37°C KRB solution. Tissues were equilibration for a period of 1 h. As previously described (Baker et al., 2018 (link)); We used a spinning-disk confocal microscope (CSU-W1 spinning disk; Yokogawa Electric Corporation) for all Ca²⁺ imaging experiments. The confocal head is connected to Nikon Eclipse FN1 microscope equipped with a 20 × 0.5 NA, 40 × 0.8 NA, and 60 × 1.0 NA CFI Fluor lens (Nikon instruments INC, NY, USA). Laser at 488 nm wavelength were directed using a Borealis system (ANDOR Technology, Belfast, UK). EMCCD Camera (Andor iXon Ultra; ANDOR Technology, Belfast, UK) was used to capture the GCaMP6f emission. Images were acquired at 33 frames per second using MetaMorph software (Molecular Devices INC, CA, USA). Nicardipine (100 nM) was used during the imaging experiments to minimize contractile artifacts.

Isolated distal colons (1.5–2.0 cm), 1.5 cm rostral to the anus, were removed and placed in a Krebs-Ringer bicarbonate solution (KRB) and opened along the mesenteric border. Inside contents were cleared and the mucosal layers were removed by sharp dissection. Tissues were isolated and pinned to the base of a Sylgard-coated dish with the serosal aspect of the colon facing down. The preparation was continuously perfused with the KRB solution at 37°C for an equilibration period of 1 hour. Preparations were then visualized and imaged using a spinning-disk confocal microscope (CSU-W1 spinning disk; Yokogawa Electric Corporation, Tokyo, Japan) mounted to an upright Eclipse FN1 microscope equipped with 40× 0.8 NA and 60× 1.0 NA CFI Fluor lens (Nikon Instruments Inc, Melville, NY) with a continuous KRB perfusion of approximately 2 mL per minute as previously described.⁸ (link)^,44 (link) The GCaMP6f Ca²⁺ indicator expressed in PICs was excited at 488 nm using a laser coupled to a Borealis system (ANDOR Technology, Belfast, UK) to increase laser intensity and uniformity. The fluorescence emission (>515 nm) was captured using a high-speed EMCCD Camera (Andor iXon Ultra; ANDOR Technology). Experiments were performed in the presence of TTX, atropine, and L-NNA to exclude effects from neurogenic, cholinergic and nitrergic pathways.