The power of light microscopes to resolve fine details has just doubled. A new technique can distinguish tiny structures inside cells, in colour and 3D, even if they are only 100 nanometres apart.

“We have opened a door to a whole new world of structures that you could not see and study before,” says Heinrich Leonhardt of the Center for Integrated Protein Science at the Ludwig Maximilians University in Munich, Germany.

The resolution achievable with light microscopy – the diffraction limit – is normally restricted to about half the wavelength of visible light, around 200 nanometres. If two objects are closer together than this, they cannot be distinguished from each other and appear as one structure.

Electron microscopy, which uses much shorter wavelengths, can visualise smaller details, but is limited to black and white images and thin or very small specimens.

Now Leonhardt’s team, along with John Sedat and colleagues at the University of California in San Francisco, US, have found a new trick to push past the diffraction limit.

Structured light
Shining structured patterns of light on specimens creates an interference pattern cast by the tiniest fine structures of the sample. This can be used to extract information about their shapes even if they cannot be visualised directly.

“It’s similar to what happens when you try to scan in a printed photo,” explains Leonhardt. “Your eye does not resolve the very fine colour dots on the paper, but the scanner does, and to your disappointment you see waves or shadows across the scanned image.”

But these interference patterns actually contain valuable information, Leonhardt realised. “With some maths and good computers we can use this to reconstruct the image,” he says.

Glowing DNA
The researchers created high-resolution views of fixed mouse cells, which had been stained with three different fluorescent colours. These labelled the DNA, the nuclear envelope and the pores through which molecules are transported into and out of the nucleus.

The new approach might make it possible to study in much greater detail how chromosomes and other subcellular components are structured in space. It could also distinguish DNA regions with active genes from those with inactive genes – an important step for understand ageing and many diseases.

Volker Westphal at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, says the new technique is promising: “Others, including our group, have broken the diffraction limit before and we can now image structures as small as 40 nanometers in 3D and even in live cells, but this work is exciting because it creates excellent images of complex subcellular structures and permits biologists to make use of the full range of fluorescent colours they like to use”.

Journal reference: Science, vol 320, p1332