Super resolution microscopy pinpoints T cell trigger

Many things are too small to be seen by the naked eye. And some are even too small to be seen through a conventional microscope.

But a super resolution fluorescence microscope is no ordinary microscope. A team at the University of New South Wales led by Associate Professor Katharina Gaus and PhD candidate David Williamson have used the only super resolution fluorescence microscope of its kind in Australia to reveal a crucial feature that enables T cells to function.

T cells, the soldiers of the immune system, need to be switched on in order to perform their protective role. The long-standing question has been: how?

Gaus and her team honed in on the particular molecular switch that spurs T cells into action. That molecule is the adaptor protein, Lat. However, how Lat triggered the required signalling was a mystery.

Using the microscope, they found that the signalling wasn’t operating through Lat on the surface of the T cells, as originally suspected, but that when T cell antigen receptors were triggered, it resulted in the recruitment of Lat from vesicles within the cell, which act as the signalling transporters.

"Previously it was thought that T-cell signalling was initiated at the cell surface in molecular clusters that formed around the activated receptor," said Gaus. "In fact, what happens is that small membrane-enclosed sacks called vesicles inside the cell travel to the receptor, pick up the signal and then leave again."

Dr Gaus said the discovery explained how the immune response could occur so quickly. "There is this rolling amplification. The signalling station is like a docking port or an airport with vesicles like planes landing and taking off. The process allows a few receptors to activate a cell and then trigger the entire immune response," she said.

Such a discovery wouldn’t have been possible using conventional microscopes – it took the light defying power of the super resolution fluorescence microscope to hone in on the fine detail of how the individual proteins interact.

"Previously you could see T cells under a microscope but you couldn't see what their individual molecules were doing," Dr Gaus said. Using the new microscope the scientists were able to image molecules as small as 10 nanometres.

PhD candidate David Williamson, whose research formed the basis of the paper, said the discovery showed what could be achieved with the new generation of super-resolution fluorescence microscopes.

"In conventional microscopy, all the target molecules are lit up at once and individual molecules become lost amongst their neighbours – it's like trying to follow a conversation in a crowd where everyone is talking at once.

"With our microscope we can make the target molecules light up one at a time and precisely determine their location while their neighbours remain dark. This 'role call' of all the target molecules means we can then build a 'super resolution' image of the sample," he said.

The next step was to pinpoint other key proteins to get a complete picture of T-cell activity and to extend the microscope to capture 3-D images with the same unprecedented resolution.

"Being able to see the behaviour and function of individual molecules in a live cell is the equivalent of seeing atoms for the first time. It could change the whole concept of molecular and cell biology," Mr Williamson said.

The microscope used by the UNSW team is a Zeiss Elyra, and is one of only five in the world. It was purchased recently using funding from an ARC Linkage Infrastructure, Equipment and Facilities (LIEF) grant.

The Elyra is a laser wide field illumination TIRF system. The system is optimised for new super-resolution techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). These techniques achieve a significant improvement of the classic resolution limit of 200 nm to 5 -10 nm resolution in x-y direction.

The system has four powerful laser lines including a 405 nm laser, a 489 nm solid state laser, a 516 nm solid state laser and a 635 nm solid state laser. Single molecules are detected with a back-illuminated EMCCD camera (Andor iXon DU-897) with a pixel size of 16 µm and a chip size of 512 x 512 pixels.

Other research team members were physicist Dr Dylan Owen, cell biologists Dr Jérémie Rossy and Dr Astrid Magenau, from the Centre for Vascular Research, and Professor Justin Gooding and Matthias Wehrmann, from UNSW’s School of Chemistry and the Australian Centre for Nanomedicine.

The research was supported by funding from the National Health and Medical Research Council, Australian Research Council and Human Frontier Science Program.

The paper was published today in the journal Nature Immunology.