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.