Professor Philipp Kukura shines new light on microscopic molecules
The ability to observe interactions between molecules on a nanometre scale is invaluable to science, yielding the kind of knowledge that, among other things, is essential to the development of new drugs. Yet this also represents a challenge; individual molecules are often just a few billionths of a metre in size, necessitating the use of powerful microscopes. Professor Philipp Kukura, Fellow in Physical Chemistry at Exeter College, has played a leading role in developing a new microscopy technology that looks set to offer powerful advances.
Professor Kukura and his team collaborated with researchers in Germany, Sweden, Switzerland and the US to develop the new technique, which works by detecting light scattering instead of the conventional approach of detecting fluorescence. Their research was published in the 27 April edition of Science. Professor Kukura commented: ‘This research has emerged from a decade of work which involved making an ever more sensitive light microscope.’ By 2014 researchers were able to use light scattering to visualise individual proteins, although the quality of the images produced still lagged behind those from conventional fluorescent microscopes.
Commenting on the existing technology, Professor Kukura said: ‘Single molecules have been observed in light microscopes since the late 1980s, but essentially all [current] optical techniques rely on fluorescence, which is the emission of light by a material after being “excited” by the absorption of electromagnetic radiation. As immensely powerful as that is, it is not universal.’
Fluorescent microscopy often requires the addition of a chemical marker to make molecules emit fluorescent light, which degrades over time. Once this has been done, the electromagnetic radiation needed to get the molecules to emit fluorescent light can also damage cells, hindering scientists’ ability to study the molecules within. These difficulties prompted researchers to investigate a different approach. Instead of observing fluorescence, they would identify molecules by observing the light these molecules scattered, without damaging the samples. Light scattering occurs when molecules or particles deflect light, a phenomenon responsible for, among other things, the blue colour of the sky. By observing the light scattering, the researchers have shown that they can actually measure the mass of the molecules that produced the scattering. This strategy has been named interferometric scattering mass spectrometry, or iSCAMS.
Advances in recent years closed the gap between fluorescence and light scattering microscopy techniques, opening the way to practical applications of the technology co-developed by Professor Kukura and his team. Professor Justin Benesch of Oxford’s Department of Chemistry, an expert in mass measurement and co-author of the work, highlighted the benefits of iSCAMS: ‘Our approach is therefore broadly applicable and, unlike traditional single-molecule microscopy, does not rely on the addition of labels to make molecules visible.’ Potential applications include studies of protein-protein interactions and even diagnostics.
Compared to existing technologies iSCAMS offers greater practicality. Professor Kukura said: ‘It measures mass with an accuracy close to that of state-of-the-art mass spectrometry, which is expensive and operates in vacuum – not necessarily representative of biological systems – whereas iSCAMS does so with only a very small volume of sample and works in essentially any aqueous environment.’
As most physiological and pathological processes occur in solution, being able to observe molecules interact in aqueous environments has potential to give invaluable and realistic insights to scientists. Professor Benesch added: ‘This enables a lot of the things that researchers want to quantify: do certain molecules interact and, if yes, how tightly? What is the composition of the protein in terms of how many pieces it contains, and how does it grow or fall apart?’
As further acknowledgement of the technology’s significance, Professor Kukura was recently named a finalist in the 2018 Blavatnik Awards for Young Scientists in the United Kingdom, a scheme established to help young scientists early in their careers when the need for their work to receive recognition and funding is often greatest. Professor Kukura was one of only nine academics to be recognised by the scheme in its inaugural year in the UK and will receive $30,000 in unrestricted funds to support his pioneering research. Speaking on his achievement, Professor Kukura said: ‘I am incredibly honoured to be part of the first cohort of Blavatnik awardees in the UK. It’s a unique acknowledgment of our science in a way that traditionally only happens much later in a scientific career, if at all, making it particularly special.’
The team is currently working on commercialising the technology so that more scientists can take advantage of its potential. It is hoped this innovative technique will revolutionise the way we study biomolecules.
The full paper, ‘Quantitative mass imaging of single biological macromolecules’, can be found in the journal Science.