AbstractCryo-electron tomography allows to observe biological structures in their native state and is increasingly used to determine the structure and ultrastructure of protein complexes, viruses, bacteria, cells, and ultimately tissues. To image samples by cryo-electron tomography and reconstruct 3D volumes, samples need to be thinner than 300nm to be sufficiently “transparent” for electrons to pass through the sample. Cells, most bacteria and tissues exceed these dimensions. Samples therefore need to be “thinned” and this can be achieved by cryo-focus ion beam (FIB) milling in a cryo-FIB-SEM. Cryo-tomograms can subsequently be acquired in a high-end cryo-TEM such as the Titan Krios. We have established a workflow for cryo-FIB milling using a Helios G4 FIB-SEM equipped with a Leica VCT500 cryo-stage. While routinely using this technique for tissue culture cells, we have recently started to explore ways to perform cryo-tomography on tissue samples. The talk will discuss examples of cryo-tomography applied to cells as well as to zebrafish and mouse tissues.microstructural features. Further, oxygen PFIB works very efferently with polymeric and biological samples producing smooth, curtain-free surfaces allowing dozen of nm thick material removal in automated serial sectioning. The samples and RoI coordinates from µCT data are tracked in the PFIB-SEM platform by a navigation software where µCT information from a visualization package and SEM images are co-registered. Finally, in the nano scale a TEM data is pinpointed from PFIB prepared lamella and imaged with a TEM microscope.
A/Prof Georg Ramm
Head Monash Ramaciotti Centre for Cryo-EM, Head Advanced Cellular Imaging Lab, Biomedicine Discovery Institute, Monash University
Georg Ramm trained as a microscopist, cell biologist, and biochemist during a PhD at Utrecht University in the Netherlands, and during undergraduate and diploma degrees in Germany. He established the Monash Ramaciotti Centre for Cryo-EM, which serves both cell and structural biologists with imaging expertise ranging from immuno EM, correlative light and electron microscopy, and volume imaging to single particle cryo-EM and cryo-tomography. Georg is a Chan Zuckerberg Initiative Imaging Scientist focusing on cellular cryo-EM.