Lab rotation project description
This rotation project is to build the skills required for hydrated specimen imaging and spectroscopy.
Tissue samples will be taken and prepared for fluorescent imaging of the vasculature imaging. Of particular interest is the glycocalyx a polysaccharide layer in the vessel lumen. Paired samples high pressure frozen and freeze substituted for studying molecular make up by ion induced mass spectroscopy.
The student will learn practical skills such as immuno-labelling, embedding sectioning and quantitative imaging. The project is geared towards preliminary data for the connected PhD using the new £2M ToF-SIMS machine and preparation equipment.
Figure: The glomerular filtration capillary glycocalyx. 3D TEM reconstruction into a 1.2µm by 0.73µm by 0.16µm cuboid. Glycocalyx (yellow), endothelium (blue) and podocyte (red)
Dr David Scurr, School of Pharmacy
The ability for a blood vessel to transport molecules (including proteins, chemokines and drugs) to and from tissues depends on its wall structure. One of the key structures is the endothelial glycocalyx, a polysaccharide layer that acts as a molecular filter. The organisational structure is key to the function of this filter and can be disrupted in many vascular pathologies; however, which specific aspects of this structure are changed remains unknown.
Here we will test the general hypothesis:
The amount and patterning of sulphur on the polysaccharide layer changes the organisational structure according to physiological vascular function.
The project is based around a key preparation technology of high pressure freezing. The technique can freeze biopsies without ice crystals so nanoscale microscopy and spectrometry is possible. The hydrated sample can then be sectioned and used in both the ToF-SIMS for chemical image analysis or transmission electron microscopy for structural and elemental analysis.
Aim 1: Develop stain free imaging and spectroscopy of the glycocalyx in collaboration with ongoing projects.
Aim 2: Determine how physiological variation of sulphur patterning on tissue and cell culture determines organisational structure.
Aim 3: Force specific polysaccharide knockdowns in culture (and collaborate with specific in-vivo genetic models) to determine likely mechanistic approaches to common diseases affecting organisational structure.