The accuracy of STEM mass measurements was discussed by us and by others in several reviews. Counting statistics limit both the accuracy of alignment, and the accuracy of mass measurement. The expected performance of STEM can be calculated easily from probe size, specimen geometry, detector geometry, atomic scattering cross-sections, and dose. The bright field, small angle (SA) dark field, and large angle (LA) dark field detectors each have counting efficiency close to unity. For typical specimens, we can calculate the expected mass accuracy at 10 electrons/A2: Fab fragments (50 k')a + 12%), glutamine synthetase (620 kDa + 2%), earthworm hemoglobin (3.6 MDa +0.9%), and tailess T7 particles (50 MDa +0.2%). Mass accuracy agrees well above 2%, but does not improve as expected for larger particles. We have adopted tailess T7 virus (40 MDa) as a test specimen because of its well-defined structure and stability. With this we are investigating factors affecting the accuracy of mass measurements such as: wash buffers, substrate parameters, freezing conditions and freeze drying parameters. A PCMass program views the mass profile of particles in the STEM image in comparison to models in various orientations to determine distortion or locate defects. With the availabluty of STEM3, we have begun parallel studies of the same specimens in both microscopes. Those giving clean backgrounds and homogereous particles in STEMi are transferred under vacuum to STEM3 and remeasured. In STEM3 we have more control of beam dose, specimen temperature (down to LHe temperature) and data acquisition parameters. Frozen specimens will be loaded into STEM3 and freeze dried in the stage while being observed.
Showing the most recent 10 out of 101 publications
