Figure 4. Intravital imaging of the mouse beating heart microcirculation in vivo and multiphoton imaging of the heart ex vivo.

(A–C) An in-house–designed 3D-printed stabilizer is lowered onto the beating left ventricle, allowing confocal intravital imaging in its center. Only a small surface of the beating heart has its motion reduced enough to permit imaging. (D) No BP/heart rate changes were detected using this approach as determined by photoplethysmography in both adult and aged sham and IR-injured mice. The graph presented shows BP, which remains constant even after the stabilizer is attached. (E) To ascertain whether any thromboinflammatory and vasculoprotective events imaged intravitally on the surface of the heart were also occurring in the deeper layers of the myocardium, multiphoton microscopy was used. The heart was cut in half longitudinally from the base to apex to expose the inner endocardial layer lining the left ventricle chamber. It was then placed on a specimen holder and attached to a tissue vibratome to precisely section the left ventricle wall into 4 × 300 μm thickness sections from the outermost layer closest to the epicardium, through to the inner layer closest to the endocardium. (F) Multiphoton Z-stacks were taken from all 4 layers, namely the (i) outermost layer closest to the epicardium — epi-to-mid (ii) outer myocardial layer — mid-to-mid (iii) inner myocardial layer — mid-to-mid, and (iv) innermost layer closest to the endocardium — mid-to-endo, avoiding the last section if it had “missing” myocardium due to sectioning through the actual ventricle chamber. Images from each layer were then rendered to form 3D stack images.