The application of energy-filtered electron microscopy to tomography of thick, selectively stained biological samples

We describe the use of an electron energy filter to enhance contrast and reduce chromatic aberration in the imaging of thick, selectively stained specimens. These efforts have been driven by the need for scientists investigating biological structure at scales just below the 0.3-mu m resolution achievable with a light microscope, which we term the "mesoscale," to produce data sets that represent complete models of large structures at the resolution of the electron microscope. For thick specimens, however, the resolution of electron microscopy is severely limited by chromatic aberration that results from the inability of current electron lenses to deal uniformly with beam electrons that have a distribution of energies as a result of inelastic scattering in the samples. The technique we describe is that of automated, most-probable loss (MPL) tomography. We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in the resolution of the projected images and the resulting computed volumes versus unfiltered transmission electron microscopy (TEM) methods on the JEOL JEM-3200 omega-filtered microscope. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. MPL tomography effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by improving resolution relative to unfiltered TEM; it also increases the signal-to-noise in comparison to zero-loss imaging of thick sections. The method therefore expands the utility of the intermediate voltage electron microscope (IVEM) for delivering information from specimens up to 3-mu m thick.