Going beyond the limits of light

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Super-resolution techniques have pushed back the limits of optics, becoming an essential tool in the life sciences.

Image of a cell skeleton


Four techniques that have revolutionised the field

A light beam randomly activates fluorescent markers so that only a few isolated molecules in the sample contribute to the fluorescent image, which is built piece by piece.

The object is illuminated by a predefined pattern of light beams. This results in patterns of interference in the image that are analysed by computer to generate greater detail from the image than allowed by the diffraction barrier.

Two photon
Two photons that each have half the required energy trigger fluorescence. They can penetrate more deeply into tissues, but do not deliver images that go beyond the diffraction barrier.

A beam of light focussed on a point illuminates an object and passes through a small aperture that blocks any light not coming from the focal point. This considerably increases the contrast, but does not break the diffraction barrier.

In 1873, German physicist Ernst Abbe established that there was a limit to microscopic resolution: the so-called diffraction barrier, determined by the wavelike nature of light itself. He showed that the highest resolution of a light microscope had to be less than half the wavelength of the light used. Thus, using a light microscope, it would be impossible to see two points that were less than 200 nanometres (0.0002 mm) apart.

One hundred twenty years later, another German physicist, Stefan Hell (coincidentally, his name means “light” in German), broke that barrier with his invention of the STED technology (Stimulated Emission Depletion Microscopy). His work launched the field of super-resolution microscopy, which now allows observations with 10 times greater resolution, or 20 nm – the size of a small virus or a thin cellular membrane.

Breaking the diffraction barrier

The invention is particularly valuable in biology and medicine, where optical microscopy plays a central role. Since light does not destroy sensitive samples, living tissues can be studied in real time. Nowadays, scanning electron microscopes, atomic force or tunnelling microscopes provide much higher resolution, but they require tissues to be sectioned and prepared for observation in a vacuum.

Biomedical researchers need to observe entire living cells to be able to detect potentially crucial details, such as in the cellular processes involved in memory. University of Bordeaux neurobiologist Valentin Nägerl is studying the role of the neuroanatomy of synapses in information processing, and he needs to be able to see structures that are smaller than 100 nm. “We successfully showed that a learning stimulus expands the tiny canal that connects the post-synaptic dendritic spine to the rest of the cell,” he explains. “STED microscopy made this observation possible. This technology represents a quantum leap for us.”

Schematic of fluorescence microsopy

Fluorescence microscopy. Based on this technique, two new techniques (see figures below) can reach resolutions smaller than the wavelength of light. (Illustration: Benjamin Schulte.)

To break the diffraction barrier, Hell turned to fluorescence microscopy. In this technique, the microscope does not focus directly on the object itself, but rather on fluorescent molecules attached to it. Excited by a laser beam with a well-defined wavelength, the molecules emit a fluorescent light that is detected by the microscope. However, the technique has the same resolution problem as do light microscopes: Similar details marked by the same fluorescent molecules cannot be distinguished if they are too close to one another. The emitted light overlaps and cannot be separated accurately.

Hell decided to illuminate the molecules sequentially, in order to observe very closely spaced, yet identical details. His trick was to follow the first excitation laser beam with a second, doughnut-shaped, red-shifted beam; this shuts off the fluorescence of the molecules illuminated by the first laser, whose beam was much wider (hence the technical name “stimulated emission depletion”).

In this way, only the molecules in the centre of the doughnut – whose circumference can be reduced as much as desired – emit fluorescence and are observed by the microscope. “The resolution of the microscope no longer fundamentally depends on focussing the light but on the properties of the molecules,” says Hell. Chemistry and optics were thus combined to create an alternating light-dark scan that pushed back the limits of microscopy.

Randomness yields greater detail

Schematic of STED microsopy

STED microsopy. (Illustration: Benjamin Schulte.)

In 2006, two new methods were introduced: PALM and STORM. They were also based on the “light-dark” principle, but used specific fluorescent molecules. First, a light pulse activates the molecules; then a second pulse triggers the fluorescence process. Because the activation is time-limited and random, only a few molecules – often far from one another – show up on each fluorescence image. A visualisation of the entire sample, including all the molecules very close to one another, is obtained by superposing thousands of successively recorded images.

“This technique is widely established and less expensive than STED because it requires less optical sophistication,” explains Thomas Misgeld, professor at the Institute of Cell Biology for the Nervous System at Technische Universität München. “However, its disadvantage is the superposition of images, which can be difficult for living tissues.”

In addition, all these methods have the same drawback: they do not get pictures from deeper parts of tissues. But this is exactly what is required by scientists like Misgeld, who studies lesions on spinal cord nerves following injury or multiple sclerosis. He needs to visualise such structures as mitochondria that are located in the interior of the cell.

Schematic of PALM and STORM microsopy

PALM and STORM microsopy. (Illustration: Benjamin Schulte.)

Combining methods

To solve this problem, Winfried Denk, a German biophysicist, had – already in the 1990s – developed the two-photon microscopy that provides microscopic information from deeper tissue layers and has become routine for many scientists. Instead of using a single photon to provide the energy to excite fluorescent molecules, this technique uses two photons that each have half the needed energy. One of the main advantages is that the light used can have a higher wavelength, allowing it to penetrate more deeply into tissues.

“You don’t break the diffraction barrier, but you can obtain information to a depth of one millimetre,” says Misgeld. Using a two-photon microscope, he and his colleagues have shown that certain nerves can survive a lesion if a surplus of calcium is prevented from entering the cell in the half hour following injury. The University of Bordeaux’s Nägerl is also using the two-photon technique; he has combined it with STED to obtain super-resolution images, even from deeper layers of tissue.

“The idea of separating closely positioned object details by using the different excited states of fluorescent molecules offers a large field of play for researchers,” says Hell.

These microscopy tinkerers are combining different methods and using new molecular activation and deactivation processes to obtain ever faster, higher resolution images. Hell is convinced that there is in principle no limit to how clearly one can visualise the details of living tissues or other materials with these methods – and dreams of one day using them to observe the structure of molecules themselves.

– By Uta Deffke

Technologist 02.013

See some spectacular examples of super-resolution microscopy in: Light fantastic and Fragment of cognition


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