“The revolution will not be crystallized”—so read the headline of a news feature in Nature. This feature, which was posted September 9, explained that many of the limitations of X-ray crystallography could be overcome by a newer technique, cryo-electron microscopy. But just about three weeks later, a research article appearing in Applied Physics Letters suggests that the revolution may not be frozen, either.
This article, prepared by scientists at the University of Zurich, describes a breakthrough in low-energy electron holography: the first nanometer-resolved imaging of individual tobacco mosaic virions. This feat suggests that structural biology will soon have the benefit of a non-destructive imaging tool that truly works at the single-molecule level.
Other high-resolution imaging techniques—not just X-ray crystallography and cryo-electron microscopy, but also nuclear magnetic resonance—require averaging over a vast ensemble of entities, which means that structural details of an individual biomolecule are often lost. Low-energy electron holography, however, has unique features that let it zero in on individual molecules without destroying them before data for constructing a high-resolution image can be compiled.
The details appeared September 28 in an article entitled, “Low-energy electron holographic imaging of individual tobacco mosaic virions.”
“Nondestructive structural biology of single particles has now become possible by means of low-energy electron holography,” wrote the authors. “As an example, individual tobacco mosaic virions deposited on ultraclean freestanding graphene are imaged at 1-nm resolution, revealing structural details arising from the helical arrangement of the outer protein shell of the virus.”
The authors added that since low-energy electron holography is a lens-less technique and since electrons with a deBroglie wavelength of approximately 1 Å do not impose radiation damage to biomolecules, the method has the potential for Angstrom resolution imaging of single biomolecules.
“The low-energy electron holography has two major advantages over conventional microscopy,” said Jean-Nicolas Longchamp, the primary author and a postdoctoral fellow of the Physics department at the University of Zurich. “First, the technique doesn't employ any lenses, so the resolution won't be limited by lens aberration. Second, low-energy electrons are harmless to biomolecules.”
In many conventional techniques, such as transmission electron microscopy, the possible resolution is limited by high-energy electrons' radiation damage to biological samples. Individual biomolecules are destroyed long before an image of high enough quality can be acquired. In other words, the low permissible electron dose in conventional microscopies is not sufficient to obtain high-resolution images from a single biomolecule.
However, in low-energy electron holography, the employed electron doses can be much higher—even after exposing fragile molecules like DNA or proteins to a electron dose more than five orders of magnitude higher than the critical dose in transmission electron microscopy, no radiation damage could be observed. Sufficient electron dose in low-energy electron holography makes imaging individual biomolecules at a nanometer resolution possible.
In Longchamp's experiment, the tobacco mosaic virions were deposited on a freestanding, ultraclean graphene, an atomically thin layer of carbon atoms arranged in a honeycomb lattice. The graphene substrate is similar to a glass slide in optical microscopy, which is conductive, robust, and transparent for low-energy electrons.
To obtain the high-resolution hologram, an atomically sharp tungsten tip acts as a source of a divergent beam of highly coherent electrons. When the beam hits the sample, part of the beam is scattered and the other part is not affected. Using a distant detector on the other side of the sample, the researchers recorded the sample's high-resolution hologram, a pattern resulting from the interference of the two beams.
“This is the first time to directly observe the helical structure of the unstained tobacco mosaic virus at a single-particle level,” Longchamp said. “Since low-energy electron holography is a method very sensitive to mechanical disturbance, the current nanometer resolution could be improved to angstrom (one ten billionth of a meter) or atomic resolution in the near future by improving the mechanical stability of the microscope.”
While by now single proteins have been imaged with nanometer resolution using the same technique, the researchers' next step is to image a single protein at atomic resolution—something that has never been done before.