Tcs sp5 dmi6000 cs

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.

[Ca²⁺]_i imaging was performed 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). The time series were acquired at a resolution of 512 X 512 pixels with a frequency of 2 Hz. The calcium reporter dye was excited with a 488 nm argon laser line, and the emitted fluorescence was detected with a Leica HyD hybrid detector in the 500-700 nm range (all from Leica Microsystems, Germany), as previously described (51 (link), 54 (link), 55 (link)).

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.