Optimisation of preparatory conditions for cryogenic scanning electron microscopy of peptide hydrogels

The cryogenic scanning electron microscope (cryo-SEM) is an electron microscope variant that has the potential to allow vitrified hydrated samples to be imaged in their native state. Frozen hydrated samples are further processed before being presented onto a cold microscope stage to be stabilised for imaging under a high cryogenic vacuum chamber environment. The liquid nitrogen slush (LNS) is the routine technique for processing samples into a vitrified state before imaging. According to recent findings, the process of freezing high water content samples (hydrogels) by the LNS techniques results in crystallisation, leaving hydrogel samples with crystal ice damages such as voids and cracks upon imaging. This thesis is focused on optimising the preparatory conditions for cryogenic scanning electron microscopy of hydrogels to reduce artefacts using peptide hydrogels as a case study. Substance P (SP) is an example of a well-characterised peptide hydrogel and is used as a model peptide for the optimisation process before applying other peptide hydrogels such as priscilicidin (newly designed de novo promising antimicrobial tetrapeptide under investigation) and lanreotide (a synthetic octapeptide used as a therapy in the treatment of acromegaly). After providing a review of cryo-SEM of peptide hydrogels and how optimising conditions could help to resolve artefacts for high-resolution imaging at the nanoscale (Chapter 1), technical improvements focused on minimising artefacts were established and used as a protocol to characterise the candidate peptide hydrogels. A newly adopted liquid ethane plunge (LEP) method that is capable of freezing sample volume of 0.5 µL to 1.0 µL deposited on a thick carbon-coated copper electron microscopy (EM) grid is developed.  A novel sample mount that allows insertion of the EM grid for cryo-SEM imaging of frozen samples is designed and constructed by CSIRO. A substance P hydrogel sample (10 % w/w SP in water) was prepared and processed by the optimised protocol. The resultant cryo-SEM image shows a significant reduction in cryogenic artefacts allowing a high-resolution image of the peptide nanostructures in a form of a bundle of fibrils in a network with intricating micro voids. Subsequent image processing and analysis by ImageJ and GraphPad prism estimates these bundles to be about 54 nm in diameter with the micro voids measuring about 242 nm (0.24 µm) in diameter. There were vitrification artefacts that were suspected to result from the high cryogenic stage temperature, however, its impact on the resultant sample image was inconclusive. For the priscilicidin peptide, the cryo-SEM image at high resolution shows the formation of bundles resulting from the lateral association of nanofibrils with interspaced voids. The bundles were estimated to have a diameter of about 42 nm and the diameter of interspaced voids measuring about 100 nm. The exacerbating sublimation artefacts resulted in the inconclusive analysis of the lanreotide gel by cryo-SEM. The designed optimised protocol offers a novel perspective into the imaging of high-water content peptides using a simplified protocol that can be applied in many laboratories. Overall, the results presented in this thesis contribute to enhancing current knowledge in cryo-SEM imaging and provide a promising technique that can further be enhanced to minimise cryogenic artefacts in peptide hydrogels.