Intravital imaging of cardiac tissue utilizing tissue-stabilized heart window chamber in live animal model

Aims To develop and validate an optimized intravital heart microimaging protocol using a suction-based tissue motion-stabilizing cardiac imaging window to facilitate real-time observation of dynamic cellular behaviours within cardiac tissue in live mouse models. Methods and results Intravital heart imaging was conducted using dual-mode confocal and two-photon microscopy. Mice were anesthetized, intubated, and maintained at a stable body temperature during the procedure. LysM-eGFP transgenic mice were utilized to visualize immune cell dynamics with vascular labelling by intravenous injection of anti-CD31 antibody and DiD-labelled red blood cells (RBCs). A heart imaging window chamber with a vacuum-based tissue motion stabilizer with 890–920 mbar was applied following a chest incision to expose the cardiac tissue. The suction-based heart imaging window chamber system and artificial intelligence-based motion compensation function significantly reduced motion artefacts and facilitated real-time in vivo cell analysis of immune cell and RBC trafficking, revealing a mean neutrophil movement velocity of 1.66 mm/s, which was slower compared to the RBC flow velocity of 9.22 mm/s. Intravital two-photon microscopic heart imaging enabled label-free second harmonic generation imaging of cardiac muscle structures with 820–840 nm excitation wavelength, revealing detailed biodistributions and structural variations in sarcomeres and fibrillar organization in the heart. Conclusion The optimized intravital heart imaging protocol successfully demonstrates its capability to provide high-resolution, real-time visualization of dynamic cellular activities within live cardiac tissue.