CryoTEM tomography (CET) has become a widely applied technique for studying the architecture of vitrified cells and viruses. With the advent of direct detector technology and phase plates, new levels of detail are being revealed. However, phase-contrast imaging, on which CET depends, requires a coherent signal from elastically scattered electrons for an interpretable image. For a 200-kV electron beam, the mean-free-path (MFP) for inelastic scattering is around 200 nm. Beyond that thickness, for every elastic electron providing information, there are at least three inelastically scattered electrons that cause damage to the specimen and only contribute noise to the image. Energy filtering blocks off the inelastic electrons, but at high tilts (thicker specimen), very little signal remains. Sample damage sets an upper limit for electron dose, and the need for elastically scattered electrons sets a limit for sample thickness and tilt geometry during data collection. These factors limit the quality of the reconstruction.
CryoSTEM tomography (CSTET) provides an alternative for thicker cells where phase-contrast imaging is not optimal. STEM imaging is based on incoherent signal detection, so that all electrons that pass through the specimen provide useful information. We recently showed that CSTET can provide fine detail in reconstructions from bacteria and eukaryotic cells up to one micron in thickness. In addition, we can collect simultaneously both the bright-field (BF) and the dark-field (DF) signal, from separate detectors. The BF signal is useful for morphology, and the DF signal provides information on elemental content, since the angle of scattering is highly dependent on atomic number. We were also able to collect on-the-spot EDX spectra, to confirm the identity of elements such as phosphorus in bacterial polyphosphate bodies, and calcium plus phosphorus in mitochondrial deposits from human fibroblasts. I will present our latest results and plans for future development of the CSTET technique.