Looking into Live Cells at Nanoscale Resolution

A super-high-resolution 3-D light microscope developed at the Max Planck Institute for Biophysical Chemistry will allow biologists to watch the workings of the tiniest organelles and even individual clusters of proteins in living cells. The new technology, which has a resolution of 40 nanometers, overcomes some major limitations in existing microscopy techniques and could have important applications in dissecting exactly how drugs impact cells.

“[It’s] a tour de force–a major accomplishment,” says John Sedat, a professor of biochemistry and biophysics at the University of California, San Francisco. Using the Max Planck microscope and others that are pushing nanoscale resolution, biologists will be able to watch how live cells work at an unprecedented level of detail. “It’s going to be a revolution for biology,” says Sedat, who was not involved in the research.

In recent decades, biologists have made great strides in understanding the molecular makeup of cells, but how these parts add up to functioning cells and tissues is still something of a mystery. Using light microscopes, biologists can watch living cells at relatively low resolution; using electron microscopy, they can carefully dissect dead cells.

The new microscope “allows you to optically dissect living cells,” says Stefan Hell, head of the department of nanobiophotonics at the Planck Institute, in Göttingen, Germany, who led the instrument’s development.

Researchers used the new microscope to make the first super-high-resolution light images of tiny cell organelles called mitochondria, which are crucial for cell metabolism and play a role in the aging process. One potential application is to visualize how certain cancer drugs affect the mitochondria, whose inner workings have been invisible to 3-D light microscopy. “It’s been difficult because you couldn’t see molecules binding to each other,” making it impossible to definitively name the cause of these drugs’ effects, says Maryann Fitzmaurice, a pathologist at Case Western Reserve University, in Cleveland.

Three-dimensional light microscopes work by scanning a focused spot of light through cells in three planes. The size of this spot limits the resolution of the microscope–nothing smaller than the size of the spot can be seen. Due to a fundamental property of light called the diffraction limit, focusing light down to a size smaller than half its wavelength is impossible using conventional lenses. Many parts of the cell are smaller than half the wavelength of the light used for these techniques. Other researchers have gotten around the diffraction limit in two dimensions, or with techniques that only work with a particular wavelength of light.

The Max Planck group developed a way to get around light’s fundamental limitations by using two beams instead of one. The first light beam plays the same role–and is the same spot size–as light in a conventional microscope. It moves through the cell under study, exciting fluorescently labeled molecules inside the cell to fluoresce. The second beam “sculpts” the first, says Hell, inhibiting fluorescence created by the edges of the first beam. That reduces the effective spot size to 40 to 45 nanometers in diameter.

Fitzmaurice says that molecular-resolution microscopy will improve patient care down the line. “The focus has been on molecular defects in disease, but to really understand them you’ve got to see them in the cell,” she says. She believes that nanoscale resolution microscopy will also play an important role in advancing personalized medicine. For example, scientists have identified specific biomarkers that help predict a cancer patient’s prognosis, but not all patients with a particular biomarker respond similarly to the same treatments. Using Hell’s new microscope and others to come, biologists can do the basic research needed to understand how proteins and other molecules interact and, ultimately, to identify more precise predictors of disease.

And in the future, microscopes with nanoscale resolution might be used in hospital labs to perform truly personalized medicine. Sedat says that the next level for nanoscale resolution microscopy is to develop it for imaging not only single cells but also tissues such as surgical biopsies. “I believe we’re on the precipice of some important new directions for light microscopy,” he says.