Cardiac Calcium Imaging with 3D-Printed Probe

A left thoracotomy was performed as above and a 3D-printed titanium stabilization probe with imaging window (Allan-Rahill et al., 2020 (link); Jones et al., 2018 (link)) attached to the left ventricle free-wall using tissue adhesive (Vetbond). Texas Red-conjugated 70 kDa dextran (3% in saline; Thermo Fisher Scientific #D1830) was injected retro-orbitally to identify the vasculature. A Ti:Sapphire laser (Chameleon, Coherent) with wavelength centered at 950 nm was used to excite indicator molecules and images collected using a custom multiphoton microscope equipped with four detection channels and high-speed resonant scanners running ScanImage (Pologruto et al., 2003 (link)). Emission fluorescence was detected using long-pass dichroic mirrors and bandpass filters for GCaMP8 (517/65 nm) and Texas Red (629/56). Water was placed within a rubber O-ring of the stabilization probe to allow immersion of the microscope objective (Olympus XLPlan N 25 × 1.05 NA). ECG and respiratory signals were collected while imaging z-stacks (50–100 frames; 2 µm per z-step; 30 frames/s) in 4–5 different regions in each mouse. Image reconstruction was performed as previously described (Jones et al., 2018 (link)) by indexing image lines based on their acquired position within the cardiac or respiratory cycles.