Cryo-STEM Tomography The presentation by Sharon Wolf provides an in-depth overview of Cryo-STEM tomography, highlighting its principles, advantages, and recent advancements. Sharon discusses the limitations of conventional cryo-electron tomography (cryoET), such as the need for thin samples (≤300 nm) due to inelastic scattering constraints. Cryo-STEM tomography overcomes this by leveraging amplitude contrast, enabling imaging of thicker samples (up to 900 nm) without the need for extensive sample milling. Key technological innovations, including direct electron detectors, phase plates, and sub-tomogram averaging, are explored, alongside practical applications like visualizing mitochondrial fission/fusion dynamics, identifying calcium phosphate granules in cells, and characterizing ferritin variants. The talk also covers methodological challenges, such as optimizing semi-convergence angles and dual-axis tomography for improved resolution, and introduces cutting-edge techniques like 4D STEM for elemental mapping. Primary objective of the presentation To introduce Cryo-STEM tomography as a transformative technique for high-resolution imaging of thick biological specimens, demonstrating its advantages over conventional Cryo-ET and showcasing its applications in cellular and structural biology. Presenter Sharon Wolf Key points presented Cryo-STEM tomography leverages amplitude contrast, allowing imaging of thicker samples (up to 900 nm) compared to conventional Cryo-ET (≤300 nm), which relies on phase contrast and requires thin specimens. Key technological advancements enabling Cryo-STEM tomography include direct electron detectors, phase plates, and sub-tomogram averaging for achieving near-atomic resolution of proteins in cells. Sample preparation challenges, such as the need for cryo-fluorescence correlative microscopy and focused ion beam (FIB) milling, are addressed, though FIB milling remains technically demanding and limits sample preservation. Cryo-STEM tomography utilizes incoherent detection, making it less dependent on elastically scattered electrons and more efficient in utilizing both elastically and inelastically scattered electrons for imaging. The semi-convergence angle in STEM is critical for thick samples, with lower angles providing greater depth of field and better resolution for biological specimens. Dual-axis Cryo-STEM tomography improves resolution by mitigating the missing wedge effect, as demonstrated in recent studies of mitochondrial fission sites. Cryo-STEM tomography enables elemental mapping and identification (e.g., phosphorus, calcium) through techniques like energy-dispersive X-ray spectroscopy (EDX) and tunable camera lengths. Applications of cryoSTEM tomography include characterizing mitochondrial dynamics, identifying intracellular granules (e.g., calcium phosphate), and studying ferritin variants for MRI imaging. Deconvolution techniques, such as those developed by Michael Elbaum’s group, can rescue data lost due to the missing wedge effect, improving the interpretability of tomograms. Emerging techniques like 4D STEM offer potential for high-resolution elemental mapping, though they require significant data acquisition time and careful optimization.