Optimization of STEM imaging conditions for cryo-tomography.

For three-dimensional analysis of cryo-specimens using tomography at low and medium magnifications, the use of STEM images has some important advantages over TEM. A clear understanding of the electron dose in STEM is essential for optimized cryo-microscopy so a dose estimation technique for STEM imaging has been developed. The STEM convergence angle was shown to have a large impact on the image quality, in particular on the signal-to-noise ratio in the acquired images. Importantly, there is a clear trade-off between good STEM spatial resolution in the images with a large convergence angle and the signal-to-noise ratio, which is improved by reducing the convergence angle as much as possible. Based on this and the effects of varying specimen thickness, image magnification and acceleration voltage which were also evaluated, we discuss the optimal conditions for cryo-STEM tomography of biological specimens.