Clearer than crystal: Australia's new cryo-electron microscope

At the newly opened Clive and Vera Ramaciotti Centre for Structural Cryo Electron Microscopy, located at Monash University, stands the FEI Titan Krios cryo-electron microscope (EM). It is three metres tall, weighs around one tonne, boasts a 300 kV electron gun and is the only one of its kind in Australia.

The Ramaciotti Centre and this $5 million microscope are central to the work of the ARC Centre of Excellence in Advanced Molecular Imaging (Imaging CoE), administered by Monash University, which brings together biologists, physicists and chemists from several collaborating organisations to characterise and visualise the interactions that underpin immune responses. The CoE aims to transform our understanding of the immune system and, in doing so, also transform the science of microscopy.

The Titan Krios cryo-electron microscope with Aidan Byrne (ARC), David de Krester (Monash) and James Whisstock (Monash).

Imaging CoE Director Professor James Whisstock explained that scientists “used microscopy to look at biological structures for many decades, but these have typically been lower resolution … so you could see sort of big blobs where there were big proteins”. Some have tried to solve this problem using crystallography, but Professor Whisstock soon realised there are some things that “you’re just never going to be able to crystallise”.

The answer lay in advances in computational methods, physics and engineering, led by microscope manufacturer FEI. The company’s Titan Krios cryo-EM fires high-energy electrons through a sample that’s frozen in a pool of liquid ethane at -200°C. By snap-freezing the samples, this not only holds in place the shape of molecules and the complexes they form, but does so in their natural cellular environment of water.

Some of the electrons in the microscope’s beam are deflected or absorbed by the molecules in the sample when they pass through it, and these rays can be used to create a 2D image of the sample. Multiple images obtained by passing the electron beam through hundreds of samples can then be automatically pieced together to determine the 3D shapes.

“The Titan Krios is powerful enough to resolve those intricate 3D shapes, identifying the position of individual atoms within a biological molecule and creating exquisitely detailed models including the molecules’ loops and side chains,” Professor Whisstock said. “It fills a gap, seeing things that X-ray crystallography and the synchrotron can’t see.”

The electron gun at the heart of the Titan Krios cryo-electron microscope.

Until recently, Australian researchers had been forced to join long overseas waiting lists in order to utilise this cutting-edge technology. Professor Whisstock explained that he and his colleagues felt the need to build their own electron microscopy centre for structural biologists, believing that “if we didn’t go down this path … the Australian community would be left behind, as it were, in terms of being able to access the technology”.

Now such a centre has been established, funded with support from the Ramaciotti Foundations, the Australian Research Council, Monash University, the Walter and Eliza Hall Institute of Medical Research, La Trobe University and the National Health and Medical Research Council. Not only is it home to what is said to be the most powerful biological microscope in Australia, but the cryo-EM is also “much easier to use than a standard electron microscope”, according to Professor Whisstock.

In addition to its high-quality optics, good stability and sensitive camera systems, the Krios features an auto-loader specially developed by FEI. This “allows you to load automatically 12 samples into the microscope”, Professor Whisstock said, “and switch between them, and keep the samples stable for up to eight or nine days”.

“You can go and have a cup of coffee while the Krios does all the work,” he added.

The Krios is not the only significant piece of technology in the centre, which houses five microscopes of various in total as well as other sample preparation equipment. In fact, Monash University is already home to a large range of research platforms and facilities, all of which are underpinned by the Monash eResearch Centre for the collection, storage, processing and management of research data. Professor Whisstock said the university recognises that “different platforms can help one another” - a strategy which will be significantly augmented by the new centre.

The facility has already been booked for a range of studies, one of which will be led by Professor Whisstock himself. He and his team are conducting research into perforins, which form pores in membranes of infected cells. These pores are used by the immune system as conduits for delivering large toxic molecules and nanoparticles into infected cells - and could equally be used to smuggle compounds into cells for therapeutic purposes. The team plans to make different kinds of these molecules and study the 3D structure of the pores they form under the Krios.

Other planned research projects will investigate a potential malaria drug, antibiotic resistance, mitochondria, insulin and its receptor, transcription and more. Professor Whisstock explained that the facility was set up in order to be as accessible to the science community as possible, stating, “This type of endeavour only really is successful if it is genuinely influencing a community.” Given the amount of interest the centre and its technology has already generated - and the potential impact this technology could have on Australian research - the influence of the centre is sure to be substantial.