Integrating SHG and two-color STED microscopy for super-resolved imaging of cell-matrix interactions in myocardium

Understanding cardiomyocyte-extracellular matrix (ECM) interactions at the molecular level is essential for deeper insights into their mechanical signaling function for cardiac development, homeostasis and remodeling. We report a labbuilt microscope integrating two-color STED microscopy with second harmonic generation (SHG) microscopy to investigate the detailed architecture of cardiomyocyte-ECM interactions in murine myocardium at a subdiffractive level. SHG microscopy is used to locate possible interaction sites at the cell-ECM interface through the intrinsic SHG signal generated by collagen assemblies and myosin filaments. Two-color STED microscopy is used to obtain a subdiffractive view of proteins at sites of interest registered by SHG microscopy. Because large field-of-view (FOV) STED microscopy is still challenging, with photobleaching often a major concern, imaging only SHG-registered sites is advantageous. Further, using intrinsic contrast in the study reduces the number of biomarkers for fluorescent staining and thereby the number of detection channels for fluorescent imaging, simplifying sample preparation procedures and STED microscopy architectures. For purpose of demonstration, we show images of immunostained type I collagen, type. collagen and laminin as ECM structures of interest in rat ventricular sections without counterstaining.