3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography.
OBJECTIVE
Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed.


METHODS
Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method).


RESULTS
3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method.


CONCLUSIONS
The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy.
Physiology and Pharmacology, University of Bristol, Bristol, UK. kenton.arkill@bristol.ac.uk
Kenton P
KP
Arkill
Chris R
CR
Neal
Judith M
JM
Mantell
Charles C
CC
Michel
Klaus
K
Qvortrup
Jørgen
J
Rostgaard
Dave O
DO
Bates
Carlo
C
Knupp
John M
JM
Squire
D000818:Animals; D002196:Capillaries / Q000648:ultrastructure*; D055032:Electron Microscope Tomography; D004730:Endothelium, Vascular / Q000648:ultrastructure*; D019276:Glycocalyx / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional; D046529:Microscopy, Electron, Transmission; D051381:Rats; D017208:Rats, Wistar