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The endothelial glycocalyx layer is a ~2 µm thick glycosaminoglycan rich pericellular matrix expressed on the luminal surface of vascular endothelial cells, which has implications in vessel mechanics and mechanotransduction. Despite its role in vascular physiology, no direct measurement has of yet been made of vessel glycocalyx material properties. Vaterite microviscometry is a laser tweezers based microrheological method, which has been previously utilized to measure the viscosity of linear and complex fluids under flow. This form of microrheology has until now relied on complete recollection of the forward scattered light. Here we present a novel method to extend vaterite microviscometry to relatively thick samples. We validate our method and its assumptions and measure the apparent viscosity as a function of distance from the vascular endothelium. We observe a differential response in conditions designed to preserve the EGL in comparison to those designed to collapse it., (2011 Optical Society of America)

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.