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Shedding light on the nanoworld

When proteins in a brain tumour are marked with red and green dyes, fluorescence microscopy shows considerably finer structures (right) than previous microscopes (left). © J. Bückers, D. Wildanger, L. Kastrup, R. Medda; Max Planck Institute for Biophysical Chemistry
When proteins in a brain tumour are marked with red and green dyes, fluorescence microscopy shows considerably finer structures (right) than previous microscopes (left). © J. Bückers, D. Wildanger, L. Kastrup, R. Medda; Max Planck Institute for Biophysical Chemistry.

Early cancer detection and faster mapping of DNA are just some of the potential applications of fluorescence microscopy, an imaging technique that allows us to peer into the world of individual molecules and earned its inventor Professor Stefan Hell a share of the 2014 Nobel Prize for Chemistry.

For years, scientists thought that optical microscopes - which work using light from the visible part of the spectrum - had reached the limit of their resolution. Because light diffracts around anything smaller than half its own wavelength, it was assumed that it would be impossible to clearly view anything smaller than this size, such as the contents of our cells.

The development of fluorescence microscopy changed all that. The technique involves marking molecules with fluorescent chemicals – known as fluorophores - which glow when light is applied.

Professor Hell's innovation was to illuminate the sample by training two separate lasers on it - one to make the fluorescent molecules glow and another to switch off the glow in all but a nanometre-sized area. By repeating this process across the whole sample, he could build up a comprehensive image nanometre-by-nanometre.

Cancer detection

One promising application of the technique is the early detection of cancer. Professor Jerker Widengren, from the KTH Royal Institute of Technology in Sweden has been able to identify unique features on the surface of cancer cells and devise fluorescently marked molecules to bind with them.

‘Our first objective was to identify breast and prostate tumours,’ said Prof. Widengren, who coordinated the EU-funded FLUODIAMON project, which ran from 2008 to 2012. During this project his team was able to show that the fluorescence-based approach requires minimal amounts of human tissue for an accurate diagnosis, reducing pain and the risk of infection for patients.

‘If this discovery has taught us anything, it is that one should keep an open mind.’

Professor Johan Hofkens, University of Leuven, Belgium

They are now investigating whether the technique can be used to detect the early signs of cancer from a blood sample. ‘We think that tumours may build their own blood vessels by stimulating nearby platelets,’ said Prof. Widengren. ‘Fluorescence microscopy is the first technique that can resolve the protein distribution in these cells to show us what is going on.’

He says the potential for the technology is vast. ‘The wavelength, intensity and polarisation of the light emitted by fluorophores can also provide details on the chemical microenvironment of the molecules.’

Into the cell

Thanks to its super-high resolution, fluorescence microscopy also enables us to view sub-cellular structures such as DNA. Professor Johan Hofkens at the University of Leuven in Belgium leads the FLUOROCODE project, which is using a variation of the technique to sort through DNA and understand more about how mistakes in genetic read-out could be linked to cancer.

Although genetic sequencing is getting faster and cheaper by the day, Prof. Hofkens says that assembling DNA readouts remains a painstaking task. The FLUOROCODE project, which is funded by the European Research Council (ERC), is using fluorescence microscopy to map out entire genomes before they are broken down for sequencing.

Researchers on the project have devised a highly efficient method to label specific DNA sequences in our genome with fluorescent chemicals. These fluorophores can also reveal precious information about their surroundings.

‘Food, stress and even pollution can cause molecules to bind in irregular ways with our DNA,’ said Prof. Hofkens. ‘This can dangerously affect the way in which the cell reads out its genetic code.’ He is now working with a biotech company to commercialise a diagnostics tool.

However, Prof. Hofkens says the greatest legacy of the development of fluorescence microscopy may lie not in its technological breakthroughs but in changing the mind-set of the research community. ‘If this discovery has taught us anything, it is that one should keep an open mind,’ he said. ‘I feel privileged to work with a community and funding agencies that allow young scientists to push for new ideas.’

  • Early compound microscopes with more than one lens enabled images magnified by one lens to be magnified again with another. Credit: Alan Hawk, National Museum of Health and Medicine, US

    Zaccharias Janssen is credited with inventing the first compound microscope in the Netherlands, although its precise origins are the source of some debate. Prior to its invention, single lenses were used as magnifying glasses. By combining multiples lenses in a tube, this new compound microscope enabled people to see details that weren’t visible to the human eye. Improved models followed and in the mid-19th century, Englishman Robert Hooke coined the word 'cell' to describe what he had observed using the microscope.
  • In addition to his formula for the diffraction limit of light, Ernst Abbe improved the manufacturing process of optical instruments and invented a way of illuminating microscopes. Creative Commons: Daniel Mietchen

    Ernst Abbe publishes his mathematical proof that there is a physical limit to the resolution of optical microscopes, known as the diffraction limit. According to Abbe’s formula, the maximum resolution of optical microscopes is 200 nanometres, which is half the wavelength of light and around the size of a small bacteria cell. For the next century scientists accept that they would not be able to see anything smaller than this with an optical microscope, and attention turns to developing microscopes that produce images using methods other than light, such as electrons.
  • Fluorophores absorb light of a certain wavelength and re-emit light at a longer wavelength. Creative Commons: Antipoff

    At the turn of the twentieth century, biochemists learnt to bind fluorescent tails to molecules inside biological samples. These markers, called fluorophores, can emit light after being irradiated, producing images with unprecedented contrast. Over the century improvements to fluorescence microscopes led to better resolution and contrast, but they didn’t overcome Abbe’s limit.
  • Advances in fluorescence microscopy allow us to clearly see structures in the nanodimension, opening up a new tool for doctors and biologists. © G. Donnert, S. W. Hell; Max Planck Institute for Biophysical Chemistry

    Professor Stefan Hell from the Max Planck Institute for Biophysical Chemistry in Germany images an E.coli bacterium at a never-before-seen resolution using a new way of illuminating fluorophores at the nano level. In order to circumvent the diffraction limit, he used one laser to excite the fluorescent markers and another to extinguish all but a nanometre sized area. In doing so, he extended fluorescence microscopy into the nanosphere and turned on its head the assumption that optical microscopes had reached their physical limit.
  • Dr Eric Betzig and Professor William Moerner shared the Nobel Prize with Professor Stefan Hell for separate work in devising another method of circumventing Abbe’s limit in fluorescence microscopy.

    The Nobel Prize in Chemistry is awarded to Professor Stefan Hell, Dr Eric Betzig and Professor William Moerner for taking optical microscopy into the nanodimension. Scientists can now observe the interaction of individual molecules inside cells, see how molecules create synapses between nerve cells in the brain, track proteins involved in Parkinson's, Alzheimer's and Huntington's diseases as they aggregate, follow individual proteins in fertilized eggs as these divide into embryos, and much more. Further advances could lead to 3D films of living cells.
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