Biology subjects

Seifer, S.

Publications and source records attributed to Seifer, S..

3 recordsLinked to original sources

bioRxiv2025

Azimuthal Segment Imaging in cryo-STEM Tomography

Cryo-electron microscopy is transitioning from investigation of isolated macromolecules to in situ studies bridging the realms of structural and cellular biology. Newly available detector technologies enable unconventional contrast modes with particular advantages. Here we demonstrate application of quadrant diode detectors to visualize a range of biological specimens by cryo-Scanning Transmission Electron Tomography (cryo-STET). Theoretically, we decompose coherent contrast by parallax analysis to isolate phase and amplitude contributions in specimens too thick for energy-filtered TEM. We thereby expand the cryo-STEM toolchest to parallax-filtered bright field (pBF) and parallax-filtered integrated differential phase contrast ({pi}DPC) and demonstrate their advantages in tomography using T4-bacteriophages, whole cells, and cryo-lift-out lamellae of cellular multilayers. The results show significant improvements over traditional STEM modalities in a realm where conventional wide-field transmission EM imaging methods are not applicable. The commercial availability of such detectors and the relative ease and speed of image reconstruction should make this realm accessible to the broader community in life science EM and beyond. TeaserParallax-corrected cryo-STET imaging provides coherent phase and amplitude contrast of thick biological specimens.

cell biology↗

bioRxiv2025

Shadow Montage and Cone-Beam Reconstruction in 4D-STEM Tomography

Diffraction images in a scanning transmission electron microscope (STEM) provide a real-space projection of the sample at sufficient probe defocus. These so-called shadow images can be acquired patch by patch in a 4D-STEM setup using a pixelated detector and assembled into a shadow montage. Due to parallel acquisition within each illuminated patch, an upscaled bright field (BF) image is rendered efficiently in time and with little additional computational demand compared to other STEM techniques. We show that in this shadow regime described by geometrical optics, the algorithm achieves the result of a tilt-corrected bright field image. Furthermore, the solution is equivalent to cone-beam reconstruction in a particular scenario of a scanning point illumination source in a plane. The contrast transfer is similar to that of conventional wide-field TEM, but like STEM the focus is insensitive to energy loss and objective lens chromatic aberration. By adjusting the overlap between shadow patch images in the diffraction plane, the shadow montage is synchronized to specific layers in the sample, rendering a 3D shadow volume from a single dataset. The method is also amenable to conventional tilt tomography, by adding a shadow montage or shadow volume to each tilt view prior to back-projection. This approach effectively circumvents the basic presumption of parallel-projection tomography that the depth of field must be greater than the specimen thickness.

biophysics↗

bioRxiv2024

Optimizing contrast in automated 4D-STEM cryo-tomography

4D-STEM is an emerging approach to electron microscopy. While it has been developed principally for high resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryo-microscopy 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 datasets using a segmented diode and an ultra-fast pixelated detector, demonstrating the methods using a specimen of 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 contrast, including incoherent and annular bright field, integrated center of mass (iCOM), and parallax decomposition of a simulated integrated differential phase contrast (iDPC). 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.

biophysics↗