Abstract For establishing intravital heart microimaging protocol in live mouse models, an optimized suction-based tissue motion-stabilizing cardiac imaging window chamber system has been developed to enable real-time observation of dynamic cellular behaviors within the cardiac tissue. With the assistance of AI-based motion compensation technology, this imaging approach facilitated high-quality, real-time in vivo visualization of dynamic cellular behaviors in heart tissue, including immune cell trafficking and red blood cell (RBC) flow monitoring, has been successfully analyzed in vivo. Additionally, Intravital Two-photon microscopic heart imaging enabled label-free visualization of cardiac muscle tissue by second harmonic generation (SHG) signal generated from 820-840nm two-photon laser excitation. The imaging method optimized in this study can be further investigated to identify the underlying mechanisms and develop advanced treatments related to various cardiovascular diseases.