Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics

Figure 1. Surgical approaches for intravital microscopy of the beating mouse heart. (A) The cervical explanted heart model provides the benefits of studying cardiac graft vs. host interaction [image adapted from Li et al. (66) with permission]. (B) Endoscopic, suction-stabilized imaging provides a less invasive approach and enables time-lapse imaging that reduces motion and appears to leave major histocompatibility complex class-II (MHC-II) + immune cell numbers unchanged [image reproduced from Jung et al. (33) with permission]. (C) Intrathoracic approaches enable a wider field of view of the freely beating heart in mechanically ventilated animals. Optical access to the heart is gained by a left thoracotomy in the anesthetized mouse. (D) Passive tissue stabilization is achieved by a 3D printed stainless steel probe (left) with a coverglass and reservoir for water immersion of the microscope objective. Tissue adhesive is applied to the underside of the stabilization probe prior to attachment to the left ventricle. Photographs show the probe attached to the heart (top right) and a view of the heart surface with visible coronary vessels in the window (bottom right). Image adapted from Jones…

Figure 2. Image acquisition and reconstruction techniques for the beating mouse heart in vivo. (A) Heart structure is relatively stable during diastole allowing acquisition of full image frames with no motion artifact [image adapted from Lee et al. (70) with permission]. (B–F) Line-by-line reconstruction enables volumetric reconstructions at any point within the cardiac or respiratory cycles. (B) Simultaneous recording of the electrocardiogram (ECG) and lung pressure during imaging produces image frames at a single Z-plane that are captured at various points in the cardiac and respiratory cycles. The R-wave peak of the ECG (red arrows) and inspiratory peak of the lung pressure (blue arrows) are defined as the start and end of the cardiac cycle and respiratory cycle, respectively. (C) Each 512 × 512 pixel image frame is produced by raster scanning the excitation laser in the X-direction, therefore each line has a defined position in Y, a depth in Z, and a time it was captured with respect to the cardiac cycle (cardiac time) and respiratory cycle (respiratory time). (D) Each Y, Z line in the image is associated with a phase in the cardiac and respiratory cycles (yellow spots). An image…

Figure 3. Leukocyte dynamics in the mouse heart using intravital multiphoton microscopy. (A) Intravital multiphoton imaging of explanted heart tissue after heterotopic cardiac transplantation into LysM+/GFP mice shows neutrophil (green) infiltration from the host through vasculature labeled with non-targeted 655 nm Q-dots in red [image adapted from Li et al. (66) with permission]. Time stamp is h:min:s and scale bar is 60 μm (top) and 20 μm (bottom). (B) Intravital multiphoton microscopy with cardiorespiratory-dependent image reconstruction of Cx3Cr1+/GFP mice hearts enables visualization of acute morphological changes of resident cardiac macrophages (green) in response to focal, laser-irradiation injury (yellow cross-hairs) over 1 h. Images are perspective Z-projections over 50 μm in depth and insets are single Z-slice of the region outlined in yellow, from 80% of the cardiac cycle and 50% of the respiratory cycle. (C) Intravital imaging shows increased number of macrophages in the heart following myocardial infarction compared to baseline. Vasculature is fluorescently labeled with Texas Red dextran (B–D, magenta). (D) Free-running intravital multiphoton microscopy captures intravascular…