What we can see for you

At the University of Sydney, biomedical scientist Associate Professor Filip Braet studies sinusoidal cells in the liver. To do this, he uses correlative biomolecular microscopy techniques to look deep into the structure of the cells to learn about their role in transendothelial transport and colorectal cancer.

Also at the University of Sydney, structural biologist Dr Lilian Soon studies the regulation of cancer cell motility, the ability of cancer cells to move from one part of the body to another. She is using live cell imaging techniques to learn more about the mechanisms of chemotaxis - the movement by a cell in reaction to a chemical stimulus - and chemokinesis - changes in cell speed or direction.

Up at the University of Queensland, Dr Brad Marsh uses an electron microscope with a special cryo-stage specimen handling feature to examine the three-dimensional structure of pancreatic beta cells and their role in insulin production.

And over at the University of Western Australia, Dr Peta Clode looks at the relationship between structure and function in biological tissues. She uses a high-resolution ion microprobe to study the movement of carbon, nitrogen and calcium in reef corals. She is also involved in the development and application of nanoscale secondary ion mass spectrometry in biology and biomedicine.

All of this research is assisted by the Nanostructural Analysis Network Organisation Major National Research Facility (NANO), which aims to be the peak Australian facility for nanometric analysis of the structure and chemistry of materials, be they physical, chemical or biological in nature.

With a total budget of $56 million over five years, NANO has been funded as an initiative of the Federal Government's Backing Australia's Ability program with co-investment by four state governments. Spread over five nodes - the universities of Sydney, NSW, Melbourne, Queensland and Western Australia - the combined technical power of NANO currently facilitates about 1100 projects, half of which are in the life sciences.

Electron microscopy

In addition to looking deeply into the behaviour of liver cells, Filip Braet is very much interested in sample preparation. Not something everyone would admit to, but, as he says, "it all starts with good preparation".

Braet is deputy director of the Australian Key Centre for Microscopy and Microanalysis at the University of Sydney, which is the headquarters of the NANO group. Originally from the Flemish speaking part of Belgium, Braet was enticed to Australia by the microscopy capability on offer, something he couldn't find in Europe.

His particular interest is in developing correlative techniques by combining the strengths of different instruments.

"You get very powerful capability when you start combining these instruments and using correlative techniques," he says. "You might look at the same project using a variety of platforms, which provide complementary information."

He is using fluorescence with scanning electron microscopy (SEM) to look at the structure and dynamics of hepatic sinusoidal cells. "I'm also working on combining transmission light images with confocal laser section images to build up information from multiple platforms. It is the combination of layers of information that is the trick."

Sample preparation in biology, of course, often means moisture. The development of environmental - or low vacuum - scanning electron microscopy (ESEM) has been a boon for the wetter end of science.

NANO's business development manager, Dr Miles Apperley, says the NANO has vast capability in conventional high vacuum SEMs through to variable pressure and low vacuum instruments.

"Normally in a scanning electron microscope the beam scanning across the sample has to operate in a high vacuum - when you put in a biological sample that might contain some moisture, the sample is not stable and is difficult to image," he says. "These variable pressure or ESEMs offer the opportunity of putting in these samples that contain moisture."

Apperley says the University of NSW is doing work in conjunction with cardiovascular researchers on cell cholesterol, using SEM to look at the effect of cholesterol on cell morphology. And the University of WA is using its variable pressure SEMs to look at various biomaterials, including native marsupial hair structure and biomineral formation and growth in plants and animals.

Another development is the use of cryogenic techniques with transmission electron microscopy (TEM), in which the electron beam doesn't scan across the surface as in SEM but passes through vitrified material. Sample preparation using automated cryogenic techniques has vastly improved the results obtainable nowadays, Braet says.

"The advantage is that you can image your sample in native conditions. Normally you have to prepare your sample by chemical fixation and that induces changes. By quickly freezing them you look at them in their native state."

The University of Queensland is leading the way in the area of cryoTEM and has one of the top labs in the world, Apperley says. "It's always good to have the cryo technique producing 2D images, but we want to know what's happening three-dimensionally, and that's what this instrument at UQ allows. Through cryotomography we can get 3D information about cells or features within cells with nanometer resolution."

Light and laser

NANO also has a number of light microscopy techniques available, including fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), second harmonic imaging and live cell imaging.

Second harmonic imaging is an advanced tool used to image structural proteins and look at collagen formation, cellulose and starch detection, Braet says.

"This advanced confocal technique enables you to optically section tissue or cells thereby obtaining a signal from the structure if it is centrosymetric. This means structural detail can be observed without staining and at exactly half the wavelength that you excite the molecule with."

Lilian Soon's research into cancer cell motility is using NANO's optical and laser technology, Apperley says. "This is also coupled with the development of a technique to do live cell imaging, with a tool that has been invented by Dr Soon specifically for characterising chemotaxis. This has applications in drug screening and high-throughput screening technology."

The University of Sydney also has a program looking at novel green fluorescent proteins (GFP) in corals from the Great Barrier Reef, led by Dr Anya Salih. These proteins have a wide range of properties and potential applications in biotech research and industry. One such application will be as a marker for cancer research.

X-ray microtomography

While these nanostructural and microanalysis techniques can look at details on a very fine scale, the network has instruments that can look at the broader scale as well, Apperley says.

"Another strength of NANO is our capability in micro-CT - microtomography - where we are imaging in two or three dimensions using soft X-rays. Some of the projects led by Dr Allan Jones at the University of Sydney are looking changes in structure in bones, changes in bone density associated with ageing and after injury. X-ray microtomography provides valuable information here.

"Another project is looking at modelling of dental roots, aimed at improving orthodontic treatment outcomes. Yet another project is visualising structures in the ears of a mouse, the membranous structure of the inner ear, to understand the mechanisms of balance. Using conventional techniques the skull is quite hard to image through, and yet when you dissect it or dissolve away the bone you actually disrupt the positioning of the softer tissues, so when you process your sample they are not lying in the proper position. There is always a question about it but with microtomography you are able to image in situ."

Not just the shiny bits

The establishment of NANO took significant capital investment, with each of the five nodes bringing all of their tools to the table, Apperley says. "However, part of the plan was to invest in the top tier of instrumentation to raise Australia's capability to provide nanostructural analysis. NANO has invested in four instruments to a total of nearly $16 million - what we call our four flagship instruments."

These are the Imago Local Electrode Atom Probe (LEAP) at the University of Sydney, a Nova Nanolab Dualbeam Focused Ion Beam at UNSW, the Tecnai F30 300kV cryo-transmission electron microscope (CryoTEM) at UQ and the Cameca secondary ion mass spectrometer (NanoSIMS) at UWA.

"NANO is not just about shiny bits of equipment, however," Apperley says. "We are about providing capability and solutions through the provision of instrument, expert technical support and linkages to a cohort of researchers who undertake world-class research every day. The focus of NANO is very much research services, research programs and research training.

"We run courses in general microscopy techniques, specimen preparation and operating the instruments. We run them regularly across all of the nodes and we also run customised workshops for special techniques. We also offer various postgraduate research and coursework programs and the University of Sydney offers a Master of Applied Science (Microscopy and Microanalysis)."

NANO is available to all research scientists, both in the public and commercial sector. "We offer a travel and access program - or NANO-TAP - that funds travel, accommodation and instrument charges for people to access facilities anywhere in Australia. Applications are made on-line at any time via the NANO website and an outcome is typically known within a few weeks."

TAP is particularly suited to projects that are at an early stage and enables researchers to get some preliminary data that can be used to plan and justify more comprehensive projects and applications for funding by schemes offered by the NHMRC and ARC, he says.