Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: Implications for tomography of thick biological sections

Abstract

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 atomic % osmium in a carbon matrix, it was found that for 1-μm thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF-STEM tomographic tilt series from a stained 600-nm thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF-STEM but significantly less in the BF-STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF-STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.

Nanoscale 3D cellular imaging by axial scanning transmission electron tomography

“…A significant fraction of these electrons can thus be excluded from images recorded with an axial detector, leading to an improvement in spatial resolution towards the bottom surface of thick specimens. We have quantified this unexpected improvement in resolution using Monte Carlo electron-trajectory simulations12 (Supplementary Fig. 4; Supplementary Discussion 2).…”

Optimization of STEM tomography acquisition — A comparison of convergent beam and parallel beam STEM tomography

“…Thus, information collected from the top of the sample originates from a smaller probe area than information collected from the bottom of the sample. The result is again a loss of resolution depending on sample thickness, sample density, setup of ADF detector, and initial convergence angle [18,19].…”

“…The advantages of parallel STEM illumination and its possible applications for tomography were first evaluated and described in the works of Muller et al [18] followed by others (e.g. [10,[19][20][21]). …”

Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

“…More recently, ET based on the technique of scanning transmission electron microscopy (STEM) has emerged as a promising approach to obtain 3-D reconstructions from specimens of about 1 micrometer in thickness (Aoyama et al, 2008; Hohmann-Marriott et al, 2009; Sousa et al, 2009). This ability to image micrometer-thick sections could be valuable, for example, in 3-D ultrastructural studies of eukaryotic parasites either in isolated form or interacting with an intracellular host (Rocha et al, 2006; Gadelha et al, 2009; Hohmann-Marriott et al, 2009; Hanssen et al, 2010).…”