Hcx apo l water immersion objective

Imaging was performed on a Leica TCS SP5 upright confocal system using a Leica HCX APO L water immersion objective (20×, NA 1.0). Calbryte 520 was excited by a 488 nm argon laser. Emitted fluorescence was detected and measured by a Leica HyD hybrid detector in the range of 500–700 nm with the standard mode (Leica Microsystem, Wetzlar, Germany). Frequency of imaging was set to around 20 Hz at 256 × 256 pixels. Pixel size was close to 1 µm², enabling quantification of Ca²⁺ oscillations.
The islet-containing slices that were subjected to imaging were chosen randomly to avoid biased selection from the head, neck, or tail of pancreas. Three to five slices were imaged from each mouse pancreas, and 6 or 8 mM glucose-containing, temperature-maintained (37 °C) ECS were perfused to the pancreas slice during Ca²⁺ imaging. Supraphysiological (25 µM) or the physiological (50 nM) concentrations of ACh were added in the ECS to study cholinergic effects. Time period of exposure to 8 mM glucose with or without ACh was adjusted depending on the time until the plateau activity was achieved. After the glucose stimulation, slices were perfused with substimulatory glucose (6 mM), before 8 mM glucose was reintroduced to the slices, or otherwise the experiments were discontinued.

Imaging was performed on a Leica TCS SP5 upright confocal system using a Leica HCX APO L water immersion objective (20x, NA 1.0) or Leica TCS SP5 DMI6000 CS inverted confocal system using HC PL APO water/oil immersion objective (20x, NA 0.7). Acquisition frequency was set to 20 Hz at 256 x 256 pixels, pixel size to around 1 µm². Calbryte 520 was excited by a 488 nm argon laser. Emitted fluorescence was detected and measured by a Leica HyD hybrid detector in the range of 500-700 nm with the standard or photon-counting mode (Leica Microsystems, Germany).
Before [Ca²⁺]_c imaging, pancreas tissue slices were kept at substimulatory glucose concentration (6 mM) in HBS. To avoid bias related to slices originating from different anatomic regions of pancreas, slices have been mixed and randomly picked up for imaging. After the preincubation period slices were transferred into an imaging perfusion system with 6 mM glucose in ECS and maintained at 37°C, after which ECS with physiological stimulatory (8 or 9 mM) or supraphysiological glucose concentrations (12, 16 or 20 mM) were used to stimulate [Ca²⁺]_c events. Adrenalin concentrations in the concentration range between 0.1 and 5000 nM have been used. Forskolin has been used at 100 and 500 nM concentrations.

Beta cell calcium dynamics were imaged using an upright confocal microscope system Leica TCS SP5 AOBS Tandem II with a 20X HCX APO L water immersion objective, NA 1.0, and an inverted confocal system Leica TCS SP5 DMI6000 CS with a 20X HC PL APO water/oil immersion objective, NA 0.7 (all from Leica Microsystems, Germany). A 488 nm argon laser was used to excite the fluorescent dye, and a Leica HyD hybrid detector operating in the 500–700 nm range was used to detect the fluorescence that was released (all from Leica Microsystems, Germany), as previously described [27 (link),122 (link)]. The resolution used for time series acquisition was 512 X 512 pixels with a frequency of 2–10 Hz.