Intravital two-photon microscopy allows for the analysis of single-cell dynamics within intact tissues. As it is well recognized that molecular cues that regulate leukocyte trafficking into inflammatory sites differ between various tissues, it is important to study organ-specific responses. Recently, intravital two-photon microscopy has been expanded to moving organs in the mouse such as beating hearts. Unlike previous experimental approaches to image cardiac tissue explants or isolated perfused heart preparations by two-photon microscopy, intravital imaging accounts for the mechanical force transmitted to vessels by the heartbeat and accurately assesses dynamic leukocyte behavior in the coronary vessels and myocardial tissue. Intravital two-photon imaging of beating hearts is a promising experimental tool that will help elucidate cellular and molecular immune processes that contribute to a variety of cardiovascular diseases.