Optimizing Contrast in Automated 4D STEM Cryotomography.

4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryomicroscopy in application to life science and radiation-sensitive materials where dose efficiency is of utmost importance. We present a workflow to acquire tomographic tilt series of 4D STEM data sets using a segmented diode and an ultrafast pixelated detector, demonstrating the methods using a specimen of a T4 bacteriophage. Full integration with the SerialEM platform conveniently provides all the tools for grid navigation and automation of the data collection. Scripts are provided to convert the raw data to mrc format files and further to generate a variety of modes representing both scattering and phase contrasts, including incoherent and annular bright field, integrated center of mass, and parallax decomposition of a simulated integrated differential phase contrast. Principal component analysis of virtual annular detectors proves particularly useful, and axial contrast is improved by 3D deconvolution with an optimized point spread function. Contrast optimization enables visualization of irregular features such as DNA strands and thin filaments of the phage tails, which would be lost upon averaging or imposition of an inappropriate symmetry.

Quantitative atomic cross section analysis by 4D-STEM and EELS.

We demonstrate the use of a 4-dimensional scanning transmission electron microscope (4D-STEM) to extract atomic cross section information in amorphous materials. We measure the scattering amplitudes of 200 keV electrons in several representative specimens: amorphous carbon, silica, amorphous ice of pure water, and vitrified phosphate buffer solution. Diffraction patterns are recorded by 4D-STEM with or without energy filter at the zero-loss peak. In addition, Electron Energy Loss Spectroscopy (EELS) data are acquired at several thicknesses and energies. Mixed elastic and inelastic contributions for thick samples can be decoupled based on a convolution model. Measured differential cross sections between 1 and 3 mrad are due primarily to plasmon excitations and follow precisely a 1/θ2 angular distribution. The measured intensities match Inokuti's calculations of total dipole matrix elements for discrete dipole transitions alone, i.e., transitions to bound states of the atom and not to continuum states. We describe the fundamental mechanism of plasmon excitation in insulators as a two-step interaction process with a fast electron. First, a target electron in the specimen is excited, the probability for which follows from the availability of atomic transitions, with a strong dependence on the column of the periodic table. Second, the dielectric response of the material determines the energy loss. The energy of the loss peak depends primarily on the valence electrons. Elastic scattering is dominant at higher angles, and can be fitted conveniently to 1/θ3.7 with a linear dependence on atomic number for light atoms. In order to facilitate the interpretation of 4D STEM measurements in terms of material composition, we introduce two key parameters. Zeta is an analytical equivalent of classical STEM Z-contrast, determined by the ratio of elastic to inelastic scattering coefficients, while eta is the elastic coefficient divided by thickness. The two parameters may serve for identification of basic classes of materials in biological and other amorphous organic specimens.

Synchronization of scanning probe and pixelated sensor for image-guided diffraction microscopy.

A 4-dimensional modality of a scanning transmission electron microscope (4D-STEM) acquires diffraction images formed by a coherent and focused electron beam scanning the specimen. Newly developed ultrafast detectors offer a possibility to acquire high throughput diffraction patterns at each pixel of the scan, enabling rapid tilt series acquisition for 4D-STEM tomography. Here we present a solution to the problem of synchronizing the electron probe scan with the diffraction image acquisition, and demonstrate on a fast hybrid-pixel detector camera (ARINA, DECTRIS). Image-guided tracking and autofocus corrections are handled by the freely-available microscope-control software SerialEM, in conjunction with a high angle annular dark field (HAADF) image acquired simultaneously. The open source SavvyScan system offers a versatile set of scanning patterns, operated by commercially available multi-channel acquisition and signal generator computer cards (Spectrum Instrumentation GmbH). Images are recorded only within a sub-region of the total field, so as to avoid spurious data collection during flyback and/or acceleration periods in the scan. Hence, the trigger of the fast camera follows selected pulses from the scan generator clock gated according to the chosen scan pattern. Software and protocol are provided for gating the trigger pulses via a microcontroller (ST Microelectronics ARM Cortex). We demonstrate the system on a standard replica grating and by diffraction imaging of a ferritin specimen.

Sampling theory perspective on tomographic tilt increment schemes.

Given a limited radiation exposure to be distributed over a discrete number of tilted projections in tomography, the optimal collection of information depends on the tilt increment scheme. Relying on principles of sampling theory, several tilt increment schemes can be compared and quantified. Following reasoning of Saxton, a revised scheme is offered in which the tilt angle increments Δθn are proportional to 1/cosθn. The revised scheme is preferable according to matrix analysis and simulations of geometrical optics. For thin specimens, applying a cosine sampling grid similar to Hoppe's scheme can improve the results. A realistic case is examined by Dr. Probe simulation of a scanning transmission electron microscope (STEM) for an atomic model adapted from the Ferritin protein molecule. Optimal reconstruction methods that are tested include the direct algebraic method, iterative reconstruction, and a new deconvolution-based weighted back-projection, which resembles the correction filter technique in signal recovery from sub-sampled data. A non-linear correction may be accounted for by iteration of the simulation with an ad-hoc atomic model.

ClusterAlign: A fiducial tracking and tilt series alignment tool for thick sample tomography.

Thick specimens, as encountered in cryo-scanning transmission electron tomography, offer special challenges to conventional reconstruction workflows. The visibility of features, including gold nanoparticles introduced as fiducial markers, varies strongly through the tilt series. As a result, tedious manual refinement may be required in order to produce a successful alignment. Information from highly tilted views must often be excluded to the detriment of axial resolution in the reconstruction. We introduce here an approach to tilt series alignment based on identification of fiducial particle clusters that transform coherently in rotation, essentially those that lie at similar depth. Clusters are identified by comparison of tilted views with a single untilted reference, rather than with adjacent tilts. The software, called ClusterAlign, proves robust to poor signal to noise ratio and varying visibility of the individual fiducials and is successful in carrying the alignment to the ends of the tilt series where other methods tend to fail. ClusterAlign may be used to generate a list of tracked fiducials, to align a tilt series, or to perform a complete 3D reconstruction. Tools to evaluate alignment error by projection matching are included. Execution involves no manual intervention, and adherence to standard file formats facilitates an interface with other software, particularly IMOD/etomo, tomo3d, and tomoalign.

Flexible STEM with Simultaneous Phase and Depth Contrast.

Recent advances in scanning transmission electron microscopy (STEM) have rekindled interest in multi-channel detectors and prompted the exploration of unconventional scan patterns. These emerging needs are not yet addressed by standard commercial hardware. The system described here incorporates a flexible scan generator that enables exploration of low-acceleration scan patterns, while data are recorded by a scalable eight-channel array of nonmultiplexed analog-to-digital converters. System integration with SerialEM provides a flexible route for automated acquisition protocols including tomography. Using a solid-state quadrant detector with additional annular rings, we explore the generation and detection of various STEM contrast modes. Through-focus bright-field scans relate to phase contrast, similarly to wide-field TEM. More strikingly, comparing images acquired from different off-axis detector elements reveals lateral shifts dependent on defocus. Compensation of this parallax effect leads to decomposition of integrated differential phase contrast (iDPC) to separable contributions relating to projected electric potential and to defocus. Thus, a single scan provides both a computationally refocused phase contrast image and a second image in which the signed intensity, bright or dark, represents the degree of defocus.

Toward Compositional Contrast by Cryo-STEM.

Electron microscopy (EM) is the most versatile tool for the study of matter at scales ranging from subatomic to visible. The high vacuum environment and the charged irradiation require careful stabilization of many specimens of interest. Biological samples are particularly sensitive due to their composition of light elements suspended in an aqueous medium. Early investigators developed techniques of embedding and staining with heavy metal salts for contrast enhancement. Indeed, the Nobel Prize in 1974 recognized Claude, de Duve, and Palade for establishment of the field of cell biology, largely due to their developments in separation and preservation of cellular components for electron microscopy. A decade later, cryogenic fixation was introduced. Vitrification of the water avoids the need for dehydration and provides an ideal matrix in which the organic macromolecules are suspended; the specimen represents a native state, suddenly frozen in time at temperatures below -150 °C. The low temperature maintains a low vapor pressure for the electron microscope, and the amorphous nature of the medium avoids diffraction contrast from crystalline ice. Such samples are extremely delicate, however, and cryo-EM imaging is a race for information in the face of ongoing damage by electron irradiation. Through this journey, cryo-EM enhanced the resolution scale from membranes to molecules and most recently to atoms. Cryo-EM pioneers, Dubochet, Frank, and Henderson, were awarded the Nobel Prize in 2017 for high resolution structure determination of biological macromolecules.A relatively untapped feature of cryo-EM is its preservation of composition. Nothing is added and nothing removed. Analytical spectroscopies based on electron energy loss or X-ray emission can be applied, but the very small interaction cross sections conflict with the weak exposures required to preserve sample integrity. To what extent can we interpret quantitatively the pixel intensities in images themselves? Conventional cryo-transmission electron microscopy (TEM) is limited in this respect, due to the strong dependence of the contrast transfer on defocus and the absence of contrast at low spatial frequencies.Inspiration comes largely from a different modality for cryo-tomography, using soft X-rays. Contrast depends on the difference in atomic absorption between carbon and oxygen in a region of the spectrum between their core level ionization energies, the so-called water window. Three dimensional (3D) reconstruction provides a map of the local X-ray absorption coefficient. The quantitative contrast enables the visualization of organic materials without stain and measurement of their concentration quantitatively. We asked, what aspects of the quantitative contrast might be transferred to cryo-electron microscopy?Compositional contrast is accessible in scanning transmission EM (STEM) via incoherent elastic scattering, which is sensitive to the atomic number Z. STEM can be regarded as a high energy, low angle diffraction measurement performed pixel by pixel with a weakly convergent beam. When coherent diffraction effects are absent, that is, in amorphous materials, a dark field signal measures quantitatively the flux scattered from the specimen integrated over the detector area. Learning to interpret these signals will open a new dimension in cryo-EM. This Account describes our efforts so far to introduce STEM for cryo-EM and tomography of biological specimens. We conclude with some thoughts on further developments.

Thermal inactivation scaling applied for SARS-CoV-2.

Based on a model of protein denaturation rate limited by an entropy-related barrier, we derive a simple formula for virus inactivation time as a function of temperature. Loss of protein structure is described by two reaction coordinates: conformational disorder of the polymer and wetting by the solvent. These establish a competition between conformational entropy and hydrophobic interaction favoring random coil or globular states, respectively. Based on the Landau theory of phase transition, the resulting free energy barrier is found to decrease linearly with the temperature difference T - Tm, and the inactivation rate should scale as U to the power of T - Tm. This form recalls an accepted model of thermal damage to cells in hyperthermia. For SARS-CoV-2 the value of U in Celsius units is found to be 1.32. Although the fitting of the model to measured data is practically indistinguishable from Arrhenius law with an activation energy, the entropy barrier mechanism is more suitable and could explain the pronounced sensitivity of SARS-CoV-2 to thermal damage. Accordingly, we predict the efficacy of mild fever over a period of ∼24 h in inactivating the virus.

Spatial and temporal turbulent velocity and vorticity power spectra from sound scattering.

By performing sound-scattering measurements with a detector array consisting of 62 elements in a flow between two counter-rotating disks we obtain the energy and vorticity power spectra directly in both spatial and temporal domains. Fast-accumulated statistics and a large signal-to-noise ratio allow us to get high-quality data rather effectively and to test scaling laws in details.