The glomerulus is the primary filtration unit of the kidney. It is a bundle of capillaries approximately 130μm in the Rat. The filtration barrier is a specialised capillary wall of which it is believed the glycocalyx layer forms a molecular filter for large proteins. The permeability and hydraulic conductivity of the glycocalyx depends on many factors including thickness, fibre spacing, fibre organisation, and completeness of coverage. Robust structural imaging of the glycocalyx has proved non-trivial, in part due to pairing physiological variables with micrographs. Presented here is the use of Scanning Electron Microscopy paired with Focused Ion Beam Milling (FIB-SEM) to image glomerular and peritubular glycocalyx. The tissue was harvested from a left-ventricle, oncotically balanced, perfusion fixed rat (after an injected lethal dose of 0.7 mL/kg sodium pentobarbitone). In addition to standard fixation, Lanthanum Dysprosium Glycosaminoglycan adhesion (LaDy GAGa), was added to show the endothelial glycocalyx. Sections of tissue were prepared as blocks for standard transmission electron microscopy (TEM). A section was cut from a block and imaged with TEM (Phillips 100) to use as a map. The remaining block was trimmed and coated with gold for FIB-SEM. FIB-SEM imaging was performed with a Quanta 3D (FEI) with a backscatter detector (vC-BS-ED, FEI) using an accelerating voltage of 2kV. Once an area of interest is identified then the block is tilted to 52 degrees and trenches milled with a focued ion beam (Ga) either side, a 1μm layer of Pt is put on along the expected cutting depth. Serial sections were then imaged by SEM after a 10nm layer is removed by the Ga Beam. The FIB-SEM showed the capillary wall effectively over a 4μm*4μm*3μm sample volume. Estimated imaging resolution was 25nm. The serial sections were aligned and corrected for tilt angle (FIJI,NIH). Within the XYZ volume, slit diaphragms, foot processes and fenestrations were clearly visible. The glycocalyx thickness and location could be measured but not fibre spacing. The glycocalyx appeared similar in the peritubular capillaries and in the glomerulus, though it was not stained in all vessels. The peritubular capillaries had more glycocalyx contrast. The glycocalyx was not evenly stained, there were tufts of taller regions, as well as areas of little or no coverage, including the appearance of gaps, possibly related to the underlying fenestrations. In conclusion though compared to electron tomography the resolution is not as high, FIB-SEM has a much greater sample volume, giving it a useful niche in the technical armory to investigate physiological understanding of glycocalyx.