Ex‐vivo microscopy of adult zebrafish islet calcium

Islets were isolated as described (Emfinger et al. 2017). Glass‐bottomed 35 mmol/L dishes (MatTeK) were coated with 1% agarose, and glass pipette tips were used to remove a section of agarose at the plate center, creating a well. Individual islets were transferred to wells and immersed in pH 7.4 Krebs Ringer's solution buffered with HEPES (KRBH) containing 2 mmol/L glucose. The KRBH base solution consisted of (in mmol/L): NaCl 114, KCl 4.7, MgSO₄ 1.16, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 5, and HEPES 20, with 0.1% BSA. Solutions of varying glucose concentrations were flowed into the plate chamber through lines running into and out of the chamber (Fig. 2A). Bulk islet data were captured using a Zeiss Axiovert 200M microscope equipped with a Lambda DG‐4 illumination system and EM‐CCD camera and a Till photonics microscope with PolyChrome V monochromator and cooled CCD camera in the CIMED Live Cell Imaging Core (https://research.wustl.edu/core-facilities/cmed-live-cell-imaging-core/). Time lapse images used 100 msec exposure at an interval of 500 msec. For single‐cell comparisons, high resolution images were captured using a Nikon Spinning Disk confocal microscope (a motorized Nikon Ti‐E scope equipped with PerfectFocus, a Yokagawa CSU‐X1 variable speed Nipkow spinning disk scan head, and Andor Zyla 4.2 Megapixel sCMOS camera) at the Washington University Center for Cellular Imaging (http://wucci.wustl.edu/). Images of ubiquitin‐gCAMP6s fish islets were collected on a Zeiss LSM 880 Airyscan confocal microscope equipped with two non‐descanned detectors for two‐photon imaging, also at the Washington University Center for Cellular Imaging. Time‐lapse images used 100 msec exposure at 1 sec intervals. All images were analyzed in Fiji (Schindelin et al. 2012). To correct for movement in x‐ and y‐planes, images were stack registered (using StackReg, rigid body) in Fiji before analysis. All calcium image data are presented as change in fluorescence intensity relative to baseline fluorescence intensity. Because the maximum excitability of an islet or β‐cell can vary, and the intensity of islet fluorescence can vary, glucose responses are shown normalized to the change in fluorescence in response to KCl (showing maximum excitability due to islet depolarization). For determining trace cross‐correlation and synchronicity, ROI measurements were analyzed using PeakCaller in MATLAB (Artimovich et al. 2017). The KCl response was excluded from segments in which cross‐correlation analysis was performed, to capture the responses to glucose only.