Tcs nt confocal system

For the experiments with GFP blastocyst chimaeras, embryos were aggregated, cultured in KSOM medium (Summers et al., 1995 (link)) under mineral oil in an incubator (37°C, 5% CO₂ in air) for 24 h, then transferred to fresh pre-equilibrated drops of culture medium under mineral oil in a WillCo HBSt-3522, thin glass-bottomed dish (Intracel Ltd., Royston, Herts, UK). This was placed in an environmental chamber on top of the pre-heated stage (THD 60, Linkam Scientific Instruments Ltd., Tadworth, UK) of a Leica DMIRB/E inverted confocal microscope, and the atmosphere within the chamber was maintained at 37°C, 5% CO₂ in air, as described elsewhere (Sharp et al., 2017 (link)). Time-lapse images were acquired using the Leica TCS NT confocal system, and images from fluorescein isothiocyanate (FITC) and transmitted light channels were merged (Sharp et al., 2017 (link)). The percentage contributions of tauGFP-positive cells to the inner cell mass (ICM), polar trophectoderm (pTE; overlying the ICM) and mural trophectoderm (mTE; surrounding the blastocyst cavity) were estimated in chimaeric blastocysts. Regions of GFP fluorescence and non-fluorescence were measured in single optical sections from time-lapse images of early, mid and expanded blastocyst stages, as described elsewhere (MacKay and West, 2005 (link)).

To image cAMP levels, the FRET-sensor H187 was excited at 405 nm and fluorescence was measured at 480 ± 10 nm and 530 ± 10 nm in xy-mode on a Zeiss LSM700 confocal system. To image NO levels, DAR-4M was AM-loaded into cells (10 µM DAR-4M AM, for 10 min), and its fluorescence was excited at 514 nm and emission detected at 580 ± 20 nm on a Leica TCS NT system.⁴⁷ (link) To image Ca²⁺ waves, Fluo3 was AM-loaded into myocytes (5 µM Fluo3, for 10 min), and its fluorescence was excited at 488 nm and detected >520 nm on a Leica TCS NT confocal system in xt mode. To measure pH_i, myocytes were AM-loaded with cSNARF1 (10 µM, for 10 min), and fluorescence was excited at 530 nm and detected simultaneously at 580 ± 10 nm and 640 ± 10 nm on an inverted Olympus microscope with an Orca 05G CCD Camera (Hamamatsu, Japan) and Optosplit (Cairn Research, UK).