Plan apochromat 1 3 na oil dic uv vis ir

ER Ca²⁺ was measured with genetically encoded ER Ca²⁺ sensor D1ER [17 (link)] on an inverted wide field microscope (Observer.A1, Carl Zeiss GmbH, Vienna, Austria) equipped with a 40x objective (Plan Apochromat 1,3 NA Oil DIC (UV) VIS-IR, Carl Zeiss GmbH, Vienna, Austria) and a standard CFP/YFP filter cube. D1ER was excited with 425 nm and emission collected with 505dcxr beam-splitter on two sides of the camera (CCD camera, Coolsnap Dyno, Photometrics, Tucson, AZ, USA). Data acquisition and control of the fluorescence microscope setup were performed using the NIS-Elements AR software (Nikon, Vienna, Austria). Basal D1ER emission ratio (emission 530 nm/480 nm) was recorded for 2 min, while the cells were perfused with physiological buffer followed by 8 min perfusion with physiological buffer without Ca²⁺ (138 mM NaCl, 1 mM MgCl₂, 5 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 10 mM glucose, pH adjusted to 7.4). After this, the ER Ca²⁺ was emptied by perfusing the cells with 4 µM ionomycin in Ca²⁺ free buffer. Background-subtracted emission ratio of D1ER was normalized to the minimum ratio reached by ionomycin. ER Ca²⁺ leak was quantified as the D1ER ratio drop after 8 min of perfusion with Ca²⁺ free buffer.

The FRET imaging was performed on an inverted wide-field microscope (Observer.A1, Carl Zeiss GmbH, Vienna, Austria) equipped with a 40× objective (Plan Apochromat 1,3 NA Oil DIC (UV) VIS-IR, Carl Zeiss GmbH) and a standard GFP/RFP filtercube (HC 493/574 (GFP/DsR)). Illumination of AF488 and rhodamine was performed at 470 or 561 nm excitation using a pE-2 LED-illumination system (CoolLED Ltd., Andover, UK), and emissions were collected with a beam splitter (T565lpxr) on two sides of the camera. For simultaneous measurements, AF488 and rhodamine were excited for 500 ms each at 470 and 561 nm and images were recorded with a charged-coupled device (CCD) camera (Coolsnap Dyno, Photometrics, Tucson, AZ, USA). Data acquisition and control of the fluorescence microscope setup was performed using the NIS-Elements AR software (Nikon, Vienna, Austria). A custom-made semi-automated ImageJ-macro was used to select each cell and measure the AF488 and rhodamine signals for long and short-pass fluorescence signals. The FRET signals were corrected for bleed-through and crosstalk using single-labeled AF488 and rhodamine-stained samples. To determine the static FRET, the AF488 to rhodamine FRET signals were normalized to acceptor and donor fluorescence.