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

Abstract

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.

Keywords: Endothelial surface glycocalyx; Heparan sulfate; Hyaluronic acid; STORM; Ultrastructure.

Figure 1

Schematics of the working principle and the setup of the stochastic optical reconstruction microscopy (STORM). (A) The top row shows the two fluorophores (located along the two dashed lines) activated and excited simultaneously by conventional microscopy. Due to the diffraction limit, the two fluorophores cannot be separated, resulting in a blurring image. The middle and bottom rows show the principle of STORM for the localization of a single molecule to a nanometer accuracy. The fluorophores are activated and excited not simultaneously, but sequentially to be localized and separated with each other. This technique can overcome the diffraction barrier for the conventional fluorescence microscopy. (B) A representative optical set up of STORM for the fluorophore Auto 488. (a) The optical path in activation process. The 405nm wavelength laser activates the photo-switchable fluorophore Auto 488, enabling the excitation. (b) The optical path in excitation and imaging processes. The 488nm laser excites the activated “on” fluorophore and its emission is recorded by a camera.

Figure 2

Schematic showing the major roles of the endothelial surface glycocalyx (ESG) in regulating vascular functions. As a mechanosensor and transducer to the blood flow, as a molecular sieve to main the normal vascular permeability and as a barrier to circulating cells including red blood cells, leukocytes, and tumor cells.

Figure 3

Visualization of the ESG by electron microscopy and confocal microscopy. Electron microscopic views of the ESG at a rat left ventricular myocardial capillary under control (A) and after enzymatic removal (B) [40]. Confocal microscopic views of the fluorescence conjugated anti-heparan sulfate (HS)-labeled ESG at a rat mesenteric capillary, mid-plane view (C), 3-D view (D) and the cross-sectional views along the vessel longitudinal direction (E) [49].

Figure 4

ESG components and ultrastructure visualized by STORM and by confocal microscopy (A) STORM images of the anti-heparan sulfate (HS) (green, middle panel) and anti-hyaluronic acid (HA) (red, right panel) of the ESG focusing at the bottom of a bEnd3 cell. The left panel is the overlay [21]. The color bars in the middle and right panels at the bottom row (3D side views) are the scale bars in nanometer. (B) Confocal images of the same anti-HS labeled ESG of the bEnd3 cells (mouse brain microvascular endothelial cell) [55]. Yellow line indicates the region for the STORM images.

Figure 5

Visualization of the ESG and vWF labeled-WPB by STORM. The anti-HS labeled ESG (green) and anti-vWF (red) labeled-WPB images acquired by STORM for a bEnd3 cell. The top row in (A) shows those under control and the bottom row shows those after 10 min lipopolysaccharides (LPS) treatment. The left panel is the 2-D overview of a 40μm×40μm image focusing at the bottom of an endothelial cell. An enlarged top view of a yellow box (approximately 2µm × 2µm) in the left panel is shown in the middle panel and its side view in the right panel. (B) Relative intensity of the anti-HS and anti-vWF-labeled WPBs after 10 min LPS treatment compared to the control. vWF, von Willebrand factor; WPB, Weibel-Palade body [22].