Advancing Cryo-Electron Tomography with Tilt-corrected Bright-field STEM (tcBF-STEM)

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4D scanning transmission electron microscopy (4D-STEM) phase contrast imaging has advanced rapidly with both instrumental and computational developments. Alongside progress in materials science, 4D-STEM phase contrast techniques such as ptychography and integrated differential phase contrast (iDPC) have been successfully demonstrated in biological systems using cryo-electron microscopy (cryo-EM) [1,2]. Moreover, automation has been implemented and optimized for 4D-STEM techniques, including center of mass (COM) and iDPC, for cryo-electron tomography (cryo-ET) [3]. In this study, we employ the tilt-corrected bright-field STEM (tcBF-STEM) technique, which has previously demonstrated a 3-5× improvement in dose collection efficiency compared to energy-filtered transmission electron microscopy (EFTEM) when imaging ∼500–800-nm-thick specimens in 2D projections [4]. However, whether these advantages extend to cryo-ET remains to be investigated. To address this, we apply cryo-ET with tcBF-STEM on cryo-focused ion beam (cryo-FIB)-milled mouse sperm cells. 4D-STEM scans were acquired using ARINA on a Krios microscope from -42° to 42° sample tilt with a 6° tilt step. Three representative tilt series slices from the acquired dataset are shown in Figure 1, along with the averaged 2D convergent beam electron diffraction (CBED) signal overlaid with a bright field mask for the -42° tilt. 2D tcBF-STEM images were reconstructed for each tilt to generate a tilt series, which was then aligned and reconstructed using weighted back projection with Aretomo3 [5]. Orthogonal 2D projections from the reconstructed tomogram are shown in Figure 2. To determine whether the benefits observed in 2D projection imaging persist in tomography, we compare tcBF-STEM and EFTEM tomograms of similarly prepared samples with comparable thickness. The analysis includes dose collection efficiency, contrast, and signal-to-noise ratio (SNR). This study provides insights into whether the improved performance of tcBF-STEM in 2D projection translates into advantages in cryo-ET, helping to assess its feasibility and effectiveness for dose-efficient, high-contrast cryo-ET imaging. Tilt-corrected bright-field STEM (tcBF-STEM) reconstructed tilt series of cryoFIB-milled mouse sperm cells. (Top) Low-magnification overview image of the specimen, with an indication of the region where data acquisition was performed. (Bottom) Three representative slices from the reconstructed tilt series at different tilt angles: -42° (left), 0° (middle), and 42° (right). (Inset) Averaged 2D convergent beam electron diffraction (CBED) pattern overlaid with the bright field mask from the -42° tilt. Orthogonal slices of the reconstructed tomogram from tilt-corrected bright-field STEM (tcBF-STEM) cryo-electron tomography. Slices of the tomogram viewed along the XY, XZ, and YZ planes, respectively, revealing ultrastructural details of the cryoFIB-milled mouse sperm cells. Mitochondria and microtubules are prominently visualized.

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