Applying a new microscopy technique to detect individual molecules in three dimensions.

Microtubules visualized with a new microscopy technique (right) appear sharper than when viewed using conventional microscopy (left).

The intricate molecular insides of cells are coming into focus, thanks to HHMI investigator Xiaowei Zhuang at Harvard University. Zhuang has developed a three-dimensional version of her high-resolution stochastic optical reconstruction microscopy (STORM) technique, allowing scientists to find the location of cellular molecules with better resolution than conventional light microscopy.

To get a glimpse of cells' inner workings, scientists typically tag molecules with proteins or dyes that give off fluorescence. But images of this fluorescence have a resolution that's limited to a few hundred nanometers by the diffraction of the light in all directions.

With this conventional method, “if you have a very interesting structure but it's smaller than the resolution, it just looks like a featureless dot,” says Zhuang. Multiple fluorescent molecules blur together. So she and her colleagues came up with a trick.

Zhuang's STORM technique, first described in 2006, had been used only for two-dimensional imaging until now. STORM involves tagging molecules with a fluorescent label, or fluorophore, that can be switched on and off.

Her team avoids the problem of overlapping fluorescence by using low amounts of light to switch on only a small percentage of fluorophores at once. Through the microscope, researchers can pinpoint a molecule's location by calculating where the center of each dot is.

Now, Zhuang has also developed a way to determine where the molecule is in the third dimension—by analyzing the size and shape, or blurriness, of each dot. Repeating the process many times, randomly turning on fluorophores during each iteration, can reveal the precise location of all the tagged molecules in a cell.

“These cellular images are now 10 times sharper in all three dimensions,” says Zhuang, who described the technique in the February 8, 2008, issue of Science and has used it to look at the proteins that help viruses enter cells—a process involving miniscule complexes of molecules that had never before been resolved by light microscopy.

“We can solve many problems that were previously beyond our reach, but there are still things that we can't reveal,” she says. “And the closer the resolution gets to true molecular scale, the more questions arise.”

Photo: Zhuang lab