2.6 Optical mapping of ex vivo heart preparations
After the Langendorff-perfused hearts reached steady state, contraction artefacts were mimimised using blebbistatin (10 µM). Dye loading was aided by pre-perfusion with pluronic F127 (20 % w/v in DMSO). Rh237 (1 µg/ml) and Rhod2-AM (1 µg/ml) were perfused to enable simultaneous membrane potential and Ca2+ measurements at 37 ℃.
Two 530 nm LEDs (LEDC-2001, MappingLab Ltd) were used to illuminate the heart after their emissions were bandpass filtered (wavelengths 530±20 nm) to minimize stray excitation light reaching the dyes. The fluorescence light was passed through a 550 nm long-pass filter and then a dichroic mirror with a cut off of 638 nm. Fluorescence light with wavelengths above 638 nm was passed through a 700 nm long-pass filter and then imaged by the camera for recording voltage signals. Fluorescence light below 638 nm was passed through a bandpass filter (585±40 nm) then imaged by the camera for recording calcium signals (OMS-PCIE-2002, MappingLab Ltd). The raw spatial resolution was 128-by-128 pixels, the total mapping area was 16×16 mm and the temporal resolution was 900 frames/second.
The cameras of the optical mapping system, LED lights and stimulator and ECG recording were simultaneously driven by an 8 channel TTL analog-digital converter and OMapRecord 4.0 software (MappingLab Ltd.). For the analysis of optical mapping signals and generation of isochronal maps, data were semi-automatically processed using OMapScope 5.0 software (MappingLab Ltd). In this experimental configuration, stable recordings for voltage (V m) and intracellular Ca2+ (CaT) signals could be obtained for >2 hours in preliminary experiments assessing system stability.