Fast 4D diSPIM Myography: Simultaneous Diameter Tracking and High‐Resolution 4D Fluorescent Imaging of ex vivo Pressurized Resistance‐Sized Arteries

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Understanding the role of endothelial cell (EC) Ca2+ signaling in vascular function requires the ability to simultaneously measure changes in smooth muscle contractility and EC Ca2+ signaling within microdomains with high speed and resolution. Conventional confocal microscopy modalities provide excellent speed and lateral resolution, but are limited by time-dependent photobleaching, phototoxicity, poor axial resolution, and narrow focal plane – traits that make the technique ill-suited to imaging sensitive 3D samples with high speed and fidelity. Dual Inverted Selective Plane of Illumination Microscopy (diSPIM; 3i, CO) systems allow for imaging with very low photobleaching and phototoxicity, millisecond temporal resolution, and 320 nm spatial resolution in x, y and z axes over a very large volume (330, 330, 150 μm, respectively). However, the geometry of diSPIM creates unique challenges. To overcome these issues, we engineered custom pressure myography chambers that place ex vivo pressurized arteries in the correct orientation for acquisition of high-resolution fluorescent data via diSPIM while using the inverted microscope base to continuously capture vessel diameter via transmitted light (www.vasotracker.com). The system is contained within a fully enclosed environmental chamber which allows regulation of temperature, humidity, and pH. Two servo-controlled pumps provide control of pressure and flow, and allow luminal drug delivery (Living Systems, VT). Using this system, resistance-sized mouse mesenteric arteries develop typical (20-30%) myogenic tone at 85 mmHg. Application of known EC Ca2+ signaling directed vasodilators, such as GSK1016790A (30 nM), ATP (50 μM), and ACh (10 μM), produce robust increases in local EC Ca2+signaling and vasodilation. The high spatial and temporal resolution of 4D diSPIM myography illuminates the role of EC Ca2+ signaling within EC microdomains in the context of intact ex vivo pressurized arteries under conditions of physiologic temperature, pH, pressure, tone, and flow.