Microscopy can see nanoscale processes in real time

Researchers at Warwick University, alongside colleges in Japan, developed the method — coined voltage-switching electrochemical microscopy (VSM-SECM) — which can simultaneously provide information on the physical topography of surfaces, as well as localised functional activity.

‘There’s a real opportunity now to properly probe some of these nanoscale processes. If you’re working with nanoscale objects you need nanoscale measurement devices that can really tell you how the surface is acting,’ said collaborator Prof Julie Macpherson of Warwick.

The new technique builds on the principles of scanning probe microscopy, pioneered in the 1990s, where an image of a surface is obtained by mechanically moving a probe in a ‘raster scan’ of the specimen, line by line, recording the interaction.

‘You’re not relying on a light principle anymore so your resolution is controlled by how small you can make the electrode — each pixel is basically a representation of the tip current and that tip current is telling you something about the properties of the surface,’ Macpherson said.

With electrochemical microscopy the substrate surface of interest is placed in a chemical solution and the tip is lowered extremely close to it until a faradaic current formed. This gives flux information about the processes and reactions occurring, to which the tip is pre-tuned for particular molecular species. The tip then moves to the next ‘pixel’ point building an overall image. 

The advantage is that fast processes with short-lived chemicals, such as the release of neurotransmitters at synapses, can be captured in real time under physiological conditions. Other microscopy techniques have attempted to follow living process in real time, but require fluorescent labels, which limits the range of detectable species and may also interfere with the living system.

The tips are controlled by piezoelectric actuators in the Z axis, which can infer the topographical height of surface features based on the degree of deformation in the material and therefore current change.

The Warwick team’s innovation was to develop a voltage-switching tip, which essentially allows functional and topographical information to be sampled simultaneously.

‘Obtaining the topography of the cell at the same time as monitoring its activity is vital as it enables you to pinpoint key features on the cell associated with the process of interest,’ said Macpherson.

The team also developed a novel pyrolytic carbon nanoelectrode for the tip that can be produced as small as 6nm using a CO₂ laser fabrication process.

‘The resolution of the technique is determined by the size of the electrode employed — hence here, one of the real advances is the use of carbonelectrodes down to this size,’ said Macpherson.

In the most recent series of experiments performed with the technique, the team was able to visualise the locations of proteins embedded in the membrane of a cell and also capture the release of neurotransmitter molecules at a synapse.

‘You are in real time; that current at the tip is following the dynamic release of the neurotransmitter. It doesn’t just give you a picture it can give you a very accurate concentration of the chemical being released,’ Macpherson said.

As well as the obvious biological applications the technique could find a use for studying physical processes. For example, corrosive processes are thought to be driven by the activity of hotspots, and so it would be useful to locate these and better understand them.

It could also help optimise nanoparticles used in photocatalysis for applications such as artificial photosynthesis for energy generation or production of hydrogen for next-generation fuel.