10 Nov 2014

Mavericks who broke the limits on microscopy

The scientists who devised ‘nanoscopy’ techniques to examine the internal workings of living cells have won this year’s Nobel prize for Chemistry. Elizabeth Finkel looks at their achievement.

Confocal imaging, left, was state-of-the-art before STED microscopy. STED can resolve proteins such as clathrin, pictured here, showing the respective resolutions.Credit: Leica Microsystems

It’s fitting that a Nobel prize be given for breaking the limits – which is what this year’s Chemistry prize winners have done. For two centuries light microscopes could not resolve anything smaller than 200 nanometres across. At that limit it was possible to see the clear outlines of a cell – but the internal goings-on remained a blur.

But over the last 14 years three mavericks have broken that limit. They have zeroed in on enzymes working inside cells, the cargo jittering along tracks in nerve cells, and the connections forming between brain cells. They have peered at the chemistry of life. The winners were Eric Betzig from the Howard Hughes Medical Institute in Maryland, known as “Janelia”, Stefan Hell from the Max Planck Institute for Biophysical Chemistry in Germany and William Moerner from Stanford University in California.

While electron microscopes had already allowed us to look deep into cells, the sample preparation invariably killed the cell. But the Nobel-winning light microscopy, known as “optical nanoscopy”, works on living cells, and that’s “paradigm shifting,” says Gerry Rubin, director of Janelia.

Rubin compares the shift in capability to watching five minutes of video of a football game versus five still images. “Nanoscopy allows us to see the rules of the game,” he says.

At the end of the 19th century, Ernst Abbe defined the limit for optical microscope resolution to roughly half the wavelength of light, about 0.2 micrometre. This meant that scientists could distinguish whole cells, as well as some parts of the cell called organelles. However, they would never be able to discern something as small as a normal-sized virus or single proteins.Credit: Johan Jarnestad/The Royal Swedish Academy of Sciences

In 1873 German physicist Ernst Abbe realised why the goings-on inside cells were a blur under a microscope – 200 nanometres is half the wavelength of light. When two objects are spaced at less than this distance from each other the light waves bouncing off them interfere with each other like ripple patterns from two pebbles thrown close together in a pond. This realisation convinced microscopists that no matter how finely they ground their lenses they would never see objects less than half the wavelength of light. It became known as the Abbe limit.

To get around that limit the prize winners figured out how to stop light waves from nearby objects interfering with each other. Their strategy was to measure the light signals one at a time. They did this by attaching fluorescent tags to proteins of interest inside cells. These tags can be triggered to blink on and off, theoretically making it possible to register the signal from one object at a time.

From that basic idea, the researchers developed two different strategies.


Hell’s technique, first developed in 2000, is called stimulated emission depletion (STED) microscopy. It involves “stimulating” the fluorescent tags on proteins to glow with one type of light beam while simultaneously zapping them with a second “depleting” beam that turns them off again. The trick is that the depleting beam is manipulated to create a doughnut shape with a sub-200-nanometre-sized hole in its centre. Only the fluorescent proteins inside this tiny hole remain lit up. The technique has made it possible to resolve objects a mere 20 nanometres apart, such as the tiny packages transported along the length of nerve cells.

The other method muffles the signal from interfering neighbours by stimulating them very gently – somewhat like calming a noisy crowd by giving each person a turn to speak rather than all shouting at once. Betzig, who spent several years at Bell Labs, and Moerner who was previously at IBM’s Almaden research centre, developed similar methods independently. Their trick is to zap the cells using low intensities of light, so that only a fraction of the fluorescently-tagged proteins inside the cell light up at any one time. Because the lit-up proteins are usually more than 200 nanometres apart their light emissions don’t interfere, so the microscope can get a precise fix on each individual protein. By repeating the process thousands of times, they are able to build up entire images of cells’ insides. In 2006 Betzig, who actually began his work on the microscope in his living room (he’d had a 10 year break from research before being recruited by Janelia), revealed proteins in action, including a single viral protein inside a cell.

“We’re seeing new things everywhere we look. Nanoscale imaging will be the major enabling technology for the next 30-50 years,” says Rubin.

Elizabeth Finkel is editor-in-chief of COSMOS.