A man and his microscope

The Centenary Institute’s executive director, Professor Mathew Vadas, affectionately calls it “The Beast”, a two-photon microscope with a few added extras that the institute purchased for close to a million dollars last year.

The Beast, Vadas says, allows researchers to see cellular and molecular activity at an unprecedented level of clarity and will provide a remarkable insight into disease processes, in particular into cancer.

Accompanying the microscope is one of the world’s leading specialists in using multiphoton microscopy to view immune responses in real time, in vivo. Professor Wolfgang Weninger trained originally in dermatology in his native Austria, and as well as heading the Immune Imaging Group at the Centenary, has a co-appointment as professor of dermatology at the University of Sydney, where he teaches at the central clinical school and sees patients at the Royal Prince Alfred Hospital.

Also accompanying Weninger to Sydney is the core of his team from his previous position, at the Wistar Institute, a US National Cancer Institute-designated cancer centre based at the University of Pennsylvania. Together, this team is working in a number of areas, including imaging T and B cell activity in tumours, the innate immune response to influenza infection, and a model of skin infection, particularly by Leishmania major.

Members include Dr Lois Cavanagh, an immunologist and senior scientist in the group, who is working on the influenza model; Dr Lai Guan Ng, who is working on the skin infection models; Dr Paulus Mrass, on the tumour model; and Dr Nicholas Haass, a dermatologist working on the melanoma model.

“What we are interested in is the relationship between cancer and the immune system,” Weninger says. “Most tumours have developed strategies to evade the immune system, so we must determine how the immune system works and under what circumstances tumour cells evade the immune system.”

One ground-breaking study Weninger and colleagues have completed involves using the two-photon microscope to investigate how cytotoxic T cells navigate within tumours and also how the T cells interact with the tumour cells themselves.

The team has shown that tumour-infiltrating T lymphocytes (TILs) follow random migratory paths, and that their migratory properties depend on signals from the T-cell receptor. Using green fluorescent protein-reporter transgenic mice, the team were able to show in real time the intricate morphology of the T cells and how they seem to hug extracellular matrix fibres as guides.

“The tumour doesn’t only consist of the tumour cells – there’s a lot of different components,” Weninger says. “There are the extracellular matrix fibres and the stroma compartments, which keep the tumour together. And lots of blood vessels of course.

“There is a continuous interplay between these components and the tumour cells. What we are aiming to do is to visualise all of these components at the same time in an intact tumour, in real time, in order to determine how they interact. This hasn’t been possible until a few years ago when multiphoton microscopy was developed.”

The team has also been able to show how a strong anti-tumour response occurs. “The tumour cells are attacked by multiple T cells and we see that in real time now. We can also see how tumour cells die. We can see a T cell attach and how over time the tumour cell disintegrates – it loses its morphology and in the end is dead. I believe we are the first ones to ever show how a tumour cell dies in vivo, inside an intact tumour.”

The microscope purchased by the Centenary Institute has a couple of differences from other multiphoton microscopes in Australia. It has an imaging mode that uses multiple laser beams, allowing moving objects and biological process to be viewed. It also has an optical parametric oscillator (OPO), which produces longer wavelengths of light, allowing it to see deeper into living tissue.

“The key point is that it uses infrared laser light,” Weninger says. “Infrared laser light penetrates tissue much deeper and better than visible laser light, which means we can see into intact tissues up to about half a millimetre. That doesn’t sound very much but in the world of microscopy it is an entire new universe.”

With multiphoton microscopy, some of the problems with traditional confocal microscopy are also overcome. “The excitation is limited to a very tiny spot,” he says. “In confocal microscopy there is a lot of photo-damage and photo-bleaching. We can avoid that with the two-photon technology, because the excitation is limited to the focal point.

“We can now image for long periods of time, up to some hours. Another technical feature is second harmonic generation, where you can visualise the structural elements of the extracellular tissue, and this is neat for our purposes.”

Weninger says the big implications of this research are that it is now possible to define at the basic level the molecular cues of the interactions between the immune system and tumour cells. And this, hopefully, will help develop improved immunotherapeutic strategies for cancer, and to directly test the effects of cancer therapeutics within living tissues.

“And finally we hope to develop novel diagnostic methods,” he says. “A good example is in my field of the skin, for melanoma. We hope that we can use the two-photon technology to find out structural algorithms that may be able to distinguish between the good and the bad lesion, which is still a big problem in the clinic.”