Intracellular Calcium Imaging of Islets

The microfluidic device was secured onto the stage of a Ti-S inverted microscope (Nikon Instruments, Inc., Melville, NY). A Lambda XL lamp fitted with a shutter and filter wheel (Sutter Instruments, Novato, CA) containing appropriate emission filters was used to excite intracellular Fura PE3-AM at 340 and 380 nm. Fluorescence images were collected with a CCD camera (Cascade, Photometrics, Tucson, AZ) controlled by Nikon NIS Elements software (Nikon) every 20 s after a 150 ms exposure (S1 Fig). The ratio of fluorescence emission at 520 nm after the 340 nm excitation to that after 380 nm, F₃₄₀/F₃₈₀, was used to calculate [Ca²⁺]_i for each islet using calibration constants [50 (link),51 (link)] that were calculated previously using a Ca²⁺ calibration kit [52 (link)].