Fluorescent super-resolution and electron microscopy have proven to be superb techniques for the three dimensional imaging of cells, although they both possess specific disadvantages. Producing a three dimensional image using electron microscopy involves studying individual sections and then processing the information to construct a model. Such a process is laborious and can take days. Fluorescent microscopy has the advantages of being fast and high-resolution, although it is limited to revealing features which have been enhanced using molecular markers.
Soft x-ray tomography has recently come to prominence, as it can produce images of entire cells, quickly and without staining. Researchers at Helmholtz-Zentrum Berlin have now developed a new x-ray microscope which can perform tomography of an entire cell in one step [Schneider et al., Nat Meth, (2010) doi:10.1038/nmeth.1533].
X-ray microscopy works by exploiting the difference in x-ray absorption between organic materials. By flash freezing a cell it is possible to perform tomography by exposing the cell to x-rays in a number of different orientations. The new microscope is ideally suited to performing tomography as the sample space is much larger than in previous designs, allowing the sample to be easily tilted. This is thanks to the elimination of the pinhole which is traditionally placed just before the sample. Instead a monochromator and a new capillary condenser supply the monochromatic light.
Unlike previous designs, which have used incoherent light, the new microscope uses partially coherent light, such that the phase between any two waves is roughly the same. Coherent light provides a higher contrast ratio, at the cost of lowering the maximum resolution. In this case the idea was to compromise by combining a partially coherent beam with a high-resolution objective. Such a microscope could potentially image fine structures better than conventional systems.
The microscope has been used to observe the ultrastructure of cells, and has detected several features that corresponding TEM measurements missed. However, Dr Gerd Schneider is quick to point out that “Our goal is not to surpass the resolution of the TEM, in fact our resolution is significantly lower”. The key to the success of the x-ray measurements was that the microscope was able to easily image three dimensional structures, which are fundamentally problematic for TEM.
The researchers emphasize that there are uses beyond biological imaging, and that they are using the microscope to study integrated circuits. Schneider explains that “we can image fully buried interconnects in IC stacks to study effects like electromigration or stress-migration”. The team is now working on improving resolution, is the hope of being able to image viruses within cells.
Stewart Bland