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 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.