Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Abstract

A variety of volume electron microscopy techniques have been developed to visualize thick biological samples. However, the resolution is limited by the sliced section thickness (>30–60 nm). To preserve biological samples in a hydrated state, cryo-focused ion beam scanning electron microscopy has been developed, providing nm resolutions. However, this method is time-consuming, requiring 15–20 h to image a 10 μm thick sample with an 8 nm slice thickness. There is a pressing need for a method that allows the rapid and efficient study of thick biological samples while maintaining nanoscale resolution. The remarkable ability of mega-electron-volt (MeV) electrons to penetrate thick biological samples, even exceeding 10 μm in thickness, while maintaining nanoscale resolution, positions MeV-STEM as a suitable microscopy tool for such applications. Our research delves into understanding the interactions between MeV electrons and frozen biological specimens through Monte Carlo simulations. Single elastic scattering, plural elastic scattering, single inelastic scattering, and plural inelastic scattering events have been simulated. The electron trajectories, the beam profile, and the intensity change of electrons in each category have been investigated. Additionally, the effects of the detector collection angle and the focal position of the electron beam were investigated. As electrons penetrated deeper into the specimen, single and plural elastic scattered electrons diminished, and plural inelastic scattered electrons became dominant, and the beam profile became wider. Even after 10 μm of the specimen, 42% of the MeV electrons were collected within 10 mrad. This confirms that MeV-STEM can be employed to study thick biological samples.

Optimize Electron Beam Energy toward In Situ Imaging of Thick Frozen Bio-Samples with Nanometer Resolution Using MeV-STEM

“…The comparisons are performed with the electron beam energies of 300 keV, 3 MeV, and 10 MeV with the standard least square method. There is a reasonably good agreement between the simulated (circles) [11] and analytically estimated (dashed line) beam sizes with the beam energy of 300 keV and sample thickness up to 1 µm (red), beam energy of 3 MeV and sample thickness up to 4 µm (blue), as well as beam energy of 10 MeV and sample thickness up to 5 µm (black), as shown in Figure 5a. Since a cross-section fixed to the direction parallel to the incoming electrons is considered in the MC simulation [11], the SB could be underestimated with the increase in the sample thickness.…”

“…A dose-limited resolution (DLR) caused by radiation damage of imaging electrons can be estimated by Equation (11) [18,20]:…”

“…We benchmarked the beam size as a function of sample thickness predicted by the analytical model via a recently implemented MC simulation code [11,[27][28][29][30][31][32] since in the study presented in this manuscript, accurately modelling inelastic scattering events in addition to elastic scattering events is extremely important, especially in such a low electron beam energy regime, ranging from a few hundred kilo-electron-volts to tens mega-electronvolts. Precisely calculating the differential scattering cross-section of electrons in amorphous ice to align with experimental observations is not so straightforward (see Section 2.1); thus, we implemented a new Monte Carlo code [11] for the purpose of precisely controlling parameters used to model inelastic scattering events in amorphous ice.…”

“…Regarding elastic scattering, the cross-section for each angle interval is the sum of the oxygen cross-section (σ O_atom ) and two times the hydrogen cross-section (σ H_atom ), as σ ice = σ O_atom + 2σ H_atom . Regarding inelastic scattering, the cross-section is 110% of the oxygen cross-section [11]. The angle interval dθ was set to 0.001 mrad for θ smaller than 0.6 mrad, 0.01 mrad for θ smaller than 6.3 mrad, 0.1 mrad for θ smaller than 67 mrad, 1 mrad for θ smaller than 600 mrad, and 10 mrad for other θ angles.…”

“…To achieve a resolution better than 10 nm for in situ imaging of large biological samples with a thickness of up to 10 µm, we implemented an analytical model based on the elastic and inelastic characteristic angles and benchmarked it by Monte Carlo (MC) simulations [11]. This model can be used to predict the transverse beam size broadening as a function of electron energy.…”