Sometimes, you have to see it...
Bettina Purfürst has seen it all.
That's an exaggeration, of course, but she has certainly seen more, and at higher resolution, than most scientists. Bettina runs the MDC's electron microscopes as head of a unit that became the institute's first core facility back in 2000. Her history with the campus goes back farther to 1988, when she arrived in Buch as a young biologist. Since then, she has devoted her career to the electron microscope.
"Electron microscopy is a technology that most groups need, but obviously each lab cannot acquire its own instrument and gain the expertise needed to operate it," Bettina says. Currently the facility has two transmission electron microscopes and serves about 80 percent of the MDC's scientific groups. It's a lot for Bettina and her two technicians to manage: preparing samples is time-consuming, hands-on work.
Specimens must be very small
The first commercial electron microscopes were built in 1939 by the company Siemens and found immediate use by allowing scientists to overcome limitations of light microscopes. The wavelength of electrons is about 100,000 times shorter than that of visible light, permitting vastly higher resolutions that can potentially reach the molecular scale. The electron beam produced by the instrument would be scattered by atoms in the air, so studies have to be carried out in a vacuum. This requires special methods of sample preparation that are carried out by Bettina and the two technicians who work with her.
The specimen must be small enough (2-3 mm) to permit its introduction into the evacuated microscopic column and thin enough to permit the transmission of electrons. One way to achieve this is a chemical fixation and plastic embedding of the samples.This leaves a block from which extremely thin slices must be cut to produce sections for examination under the microscope.
A drawback of this procedure is that the plastic embedding might distort structures in the sample or introduce artifacts. In the 1980s the field of cryo-preparations was born, with the discovery that specimens could be flash-frozen and cut in a way that didn't produce ice crystals or introduce other distortions. The MDC facility offered this method of preparation until the retirement of one of Bettina's colleagues; she is hoping that a new member of the team, to be hired soon, will fill in the gap.
Tiny grains of gold for labeling
Currently scientists at the institute are using EM mainly to carry out detailed structural studies of tissues, cells and organelles or precisely determine the locations of molecules. The latter type of study is usually based on immunolabeling, where gold grains in the range of 2-20 nm (for comparison: a human hair has a width of 80,000 nanometers) have been decorated with antibodies to detect particular proteins. In this way, even double labelings with different gold sizes are possible.
The precise view offered by EM permits scientists to carry out detailed studies of the morphology of cells and their components and the way they change after various interventions. Gene knockouts, diseases, therapies, and other events affect cellular structure, organelles, and functions in ways that help explain their effects on organisms. Scientists use EM to compare affected cells with their healthy counterparts and search for crucial differences. The facility also receives biopsies from clinics, for this type of use in research or diagnosis.
"Any tissue might be affected by a disease or intervention," Bettina says. "So when a scientist comes to the facility, they must have a precise question, particularly regarding the types of cells or tissues that are probably changed and should be investigated – in other words, EM is not a screening facility. And sometimes even the description of the healthy, wild-type tissue or organism is not easy to achieve, because one is looking for the 'needle in the haystack' – structures that are too small or rare to locate."
Another type of study carried out by the facility involves structural investigations of molecules. Oliver Daumke's lab is using EM to obtain information about the way proteins assemble into molecular machines. Erich Wanker's lab is looking at the way fibrils form during Huntington's disease: the size and shape of the fibers change under various conditions, providing insights into the stages by which they aggregate. Their structure is also affected by other molecules associated with the fibrils – Erich's group is trying to determine how other proteins influence the folding of the subunits of the fibrils into a deadly form that can't be dissolved. All these molecules can be seen in the EM in a more direct way – adhered to grids and stained in negative contrast.
Images from the instruments are captured with high-resolution cameras that scientists are increasingly using for quantitative studies. "This permits us to take ten to twenty times the number of images that we were formerly getting with film," she says. "Still, our work is not high-throughput; it is manually quite intensive and preparing the samples requires skill and dexterity."
Evaluating the results requires more: a good pair of eyes. Bettina scrolls through images from recent projects, naming what she sees: here a section of the colon, with the crypt structures that contain stem cells; here the ducts of kidney cells, there a slice of brain tissue showing demyelination, all obtained in studies of disease. All to be compared with healthy controls, in hopes of linking the molecular scale of life with its effects on higher-level structures. With new methods of cryo-preparations and modern high-end electron microscopes, these techniques currently may reach a resolution of 0.05 nm, allowing biological molecules to be examined in their natural contexts.
