-Differential phase contrast for biological specimens
The acquisition and processing of differential phase contrast (DPC) and full 4D STEM has been implemented at current cryo-STEM hardware platforms by dedicated software developments, e.g. Velox. The implementation of routine 4D-cryoSTEM data acquisition for biological single-particle samples is currently under way. Following our earlier studies on tobacco mosaic virus (TMV) (Lazic et al. 2022), we imaged a series of other biological specimens: 70S bacterial ribosomes (2 MDa), apoferritin (500 kDa) and hemoglobin (60 kDa) using integrated DPC (manuscript in preparation). While TMV (> GDa) can be routinely resolved at near-atomic resolution, the smaller ribosome is now resolved at ~8.0 resolution while apoferritin and hemoglobin 3D image reconstructions fail due to lack of sufficient contrast. The Müller-Caspary group (LMU) has further focussed on the principal characterization of DPC based on segmented ring detectors, the spatial frequency dependent contrast transfer has been studied in both simulations of vitrified proteins. Two common manuscripts with Sachse (FZJ) and Müller-Caspary (LMU) are currently in preparation to demonstrate state-of-the-art capabilities of DPC when applied to biological specimens.
-Ptychography for biological specimens
The Stahlberg group (EPFL) has focused on the implementation of low-dose 4D-STEM for proteins and tissue sections. An aberration-corrected STEM was equipped with a TVIPS scan generator, allowing to control the electron beam at highest precision, so that the focussed beam can be stepped over the specimen at very high speed in a random, pre-defined pattern. At the same time, the instrument was equipped with a DECTRIS Arina camera, allowing to record images of 192x192 pixels at 30,000 frames per second. A setup based on SerialEM allowed the semi-automated collection of 4D-STEM datasets of different proteins. The recorded, very large 4D-STEM datasets were then subjected to image reconstruction, using the py4Dstem software that is publicly available under the GPL from a group in Berkeley, USA. This has resulted in first medium-high reconstructions from 4D-STEM, published in a common publication between the Sachse, Müller-Caspary and Stahlberg groups under Küçükoglu et al., Nature Communications (2024).
Regular common measurement campaigns at the cryo-microscopes in Lausanne and Jülich have been conducted together with the LMU partners to establish two main goals. First, a full cryo-STEM workflow was established which means the automated procedure from 4D-cryo-STEM data acquisition of millions of frames from autonomously selected regions on grids with vitrified proteins under individual, optimised foci, the ptychographic reconstructions including probe retrieval and multiple 2D projections of single particles separately, particle picking and 3D reconstruction using Relion or CryoSPARC, and to refine single projections to iteratively improve the 3D model.
-Optimizing image reconstructions of low-dose
In order to fully make use of the rich 4D-cryoSTEM data, several aspects have been scrutinized by Müller-Caspary (LMU) in detail. First, for low-dose phase retrieval, the Wirtinger Flow algorithm has been introduced to 4D STEM, and a benchmarking study of the attainable resolution in dependence of the electron dose and the type of the loss function has been published (Micron 185, 103688, 2024). Second, a considerable effort has been spent to retrieve the scanning probe under low-dose conditions. The benefits have been worked out comprehensively in experiment and simulations for direct ptychography methods (SSB, WDD), and for iterative single- and inverse multislice ptychography, which we published in Applied Physics Letters 126, 081602, 2025. Finally, contemporary cryo-STEM optics is often optimised for conventional TEM by changing the optical distances such that the illuminating probe edge is free of Fresnel fringes in parallel illumination mode. These challenges could be solved by incorporating the optical characteristics of cryo-(S)TEM hardware into our ptychographic algorithms (ePIE, Wirtinger Flow), making cryo-STEM ptychography available on the widespread cryo-(S)TEM microscopes equipped with the fringe-free setup. A common publication within the consortium is currently in preparation.
