Focus: Medical Technology: Investigation of Endothelial Surface Glycocalyx Components and Ultrastructure by Single Molecule Localization Microscopy: Stochastic Optical Reconstruction Microscopy (STORM)

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs), and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in regulating vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of conventional optical and electrical microscopy, the ultrastructure of ESG, in the order of 10 to 100 nanometers, has not been revealed until recent development of a super resolution fluorescence optical microscope, Stochastic Optical Reconstruction Microscope (STORM), which is one type of single molecule localization microscopy. This short review describes how the STORM can overcome the diffraction barrier in the conventional fluorescence microscopy to identify the chemical components of the ESG at a high spatial resolution. Examples of the organized ultrastructure of the ESG on the in vitro EC monolayer revealed by the Nikon-STORM system are given as well as how its components get lost during the onset of sepsis, a systemic inflammatory syndrome induced by bacterial infection, which demonstrate that this new technique can be applied to discover the structural and molecular mechanisms at nanometer scales in the native cellular environment for the cellular functions under normal and disease conditions