Benchmarking the ideal sample thickness in cryo-EM

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

Low-Energy Electron Inelastic Mean Free Path of Graphene Measured by a Time-of-Flight Spectrometer

The detailed examination of electron scattering in solids is of crucial importance for the theory of solid-state physics, as well as for the development and diagnostics of novel materials, particularly those for micro- and nanoelectronics. Among others, an important parameter of electron scattering is the inelastic mean free path (IMFP) of electrons both in bulk materials and in thin films, including 2D crystals. The amount of IMFP data available is still not sufficient, especially for very slow electrons and for 2D crystals. This situation motivated the present study, which summarizes pilot experiments for graphene on a new device intended to acquire electron energy-loss spectra (EELS) for low landing energies. Thanks to its unique properties, such as electrical conductivity and transparency, graphene is an ideal candidate for study at very low energies in the transmission mode of an electron microscope. The EELS are acquired by means of the very low-energy electron microspectroscopy of 2D crystals, using a dedicated ultra-high vacuum scanning low-energy electron microscope equipped with a time-of-flight (ToF) velocity analyzer. In order to verify our pilot results, we also simulate the EELS by means of density functional theory (DFT) and the many-body perturbation theory. Additional DFT calculations, providing both the total density of states and the band structure, illustrate the graphene loss features. We utilize the experimental EELS data to derive IMFP values using the so-called log-ratio method.

Benchmarking ideal sample thickness in cryo-EM using MicroED

The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several EM grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion-beam scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages but the quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods.

Surface Characterization of MoS2 Atomic Layers Mechanically Exfoliated on a Si Substrate

Mo disulfide overlayers with the thickness exceeding 1.77 nm were obtained on Si substrates through mechanical exfoliation. The resulting Mo disulfide flakes were then analyzed ex situ using combination of Auger electron spectroscopy (AES), elastic-peak electron spectroscopy (EPES) and scanning electron microscopy (SEM) in order to characterize their surface chemical composition, electron transport phenomena and surface morphology. Prior to EPES measurements, the Mo disulfide surface was sputter-cleaned and amorphized by 3 kV argon ions, and the resulting S/Mo atomic ratio varied in the range 1.80–1.88, as found from AES measurements. The SEM images revealed single crystalline small-area (up to 15 μm in lateral size) Mo disulfide flakes having polygonal or near-triangular shapes. Such irregular-edged flakes exhibited high crystal quality and thickness uniformity. The inelastic mean free path (IMFP), characterizing electron transport, was evaluated from the relative EPES using Au reference material for electron energies E = 0.5–2 keV. Experimental IMFPs, λ, determined for the AES-measured surface compositions were approximated by the simple function λ = kEp, where k = 0.0289 and p = 0.946 were fitted parameters. Additionally, these IMFPs were compared with IMFPs resulting from the two methods: (i) present calculations based on the formalism of the Oswald et al. model; (ii) the predictive equation of Tanuma et al. (TPP-2M) for the measured Mo0.293S0.551C0.156 surface composition (S/Mo = 1.88), and also for stoichiometric MoS2 composition. The fitted function was found to be reasonably consistent with the measured, calculated and predicted IMFPs. We concluded that the measured IMFP value at 0.5 keV was only slightly affected by residual carbon contamination at the Mo disulfide surface.