In conventional electron tomography, three-dimensional subcellular structure is derived from a series of bright-field images that are collected over a range of tilt angles in a transmission electron microscope (TEM). However, the visibility of cellular structures in stained specimens is often limited by low contrast of features in the three-dimensional reconstruction or by the inability to identify specific protein complexes within an organelle. The high background due to unscattered electrons often limits the signal-to-noise ratio achievable in bright-field (amplitude contrast) images of stained specimens recorded close to focus. On the other hand, the background is largely eliminated by using the technique of dark-field scanning transmission electron microscopy (STEM), where the elastic scattering is collected using an annular dark-field detector. We have implemented STEM electron tomography in a 300 kV FEI Tecnai TF30 transmission electron microscope. Tilt series are acquired automatically and corrections are made for specimen drift by means of cross correlation with a reference image. Whereas the focus changes across a tilted specimen in conventional TEM tomography, the image is kept focused in STEM tomography by dynamically focusing the probe during acquisition of the scanned image. The tilt series were aligned using larger 5 or 10 nm gold fiducial particles adsorbed onto the specimen and a three-dimensional reconstruction was performed using the IMOD program (University of Colorado). STEM tomography has been tested on stained and unstained sections containing gold particles of one-nanometer diameter, which are used to label proteins. It was found that the nanogold particles could easily be detected in STEM and measurements indicated show that the signal to noise ratio is considerably enhanced relative to that obtained using conventional TEM tomography for the same applied dose. It was also found that undecagold particles (each containing 11 gold atoms) could be detected in STEM tomography of evaporated carbon films.
