Biology subjects

Elbaum, M.

Publications and source records attributed to Elbaum, M..

14 recordsLinked to original sources

bioRxiv2026

FPGA-based scanner and SerialEM server for 4D-STEM Electron Tomography

Four-dimensional scanning transmission electron microscopy (4D-STEM) enables the acquisition of diffraction patterns at every probe position in a dense array. For imaging applications this approach offers significant benefits in terms of spatial resolution and contrast enhancement. In this work, we present the development of a synchronous scan generator integrated with SerialEM software to enable automation of complex experimental protocols such as tomography. The proposed hardware functions as an interface between SerialEM, the scan controls of the microscope, a fast annular dark-field detector, and a synchronized trigger for a pixelated detector. Our previous implementation, named SavvyScan, relied on a dedicated computer equipped with a multichannel acquisition and signal-generation cards, as well as a separate microcontroller for synchronization. Here, we report a low-cost implementation based on a Red Pitaya board, utilizing direct programming of its embedded FPGA and Linux server components. We provide detailed instructions for system installation and operation, along with practical guidance for modifying the source code. System performance is validated through oscilloscope measurements and imaging of a replica grating sample. The utility of the approach is further demonstrated by generating a 3D electron tomogram of a cryogenic sample of mitochondria from a tilt series of shadow montage projections.

biophysics↗

bioRxiv2026

3dcon: tomogram denoising by deconvolution

Cryo-electron tomography is an expanding technology for the study of macromolecules, viruses, and cells. It is often applied to specimens that are too large or heterogeneous for methods based on 2D image averaging such as single particle analysis, e.g., intracellular membranes or organelles. Current practice records a tilt series of projection images in rotation. Reconstruction is normally an ill-posed mathematical problem. Particularly for the under-determined case of sparse data, discrete tilt angles, and a limited tilt range, characteristic artifacts appear in the reconstructed slices. Much of what appears as noise is in fact structural: the projection of contrast from different planes. Various schemes are employed to regularize the reconstruction, including machine-learning frameworks built on neural networks. To the extent that the noise is structural, it might be suppressed by deconvolution with a suitable kernel. This was demonstrated and has been used regularly in cryo-STEM tomography of thick specimens where the under-sampling problem is particularly acute. Here we present 3dcon as an open-source extension of the entropy-regularized deconvolution algorithm that had been adopted from fluorescence microscopy. It takes advantage of modern computing hardware for convenient and fast processing. Deconvolution is entirely algorithmic, meaning that successful processing of the data does not depend on the data itself. As such it should be robust in a wide variety of applications.

biochemistry↗

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↗

bioRxiv2025

Refractive index as an indicator for dynamic protein condensation in cell nuclei

Protein condensation is the basis for formation of membrane-less organelles in the cell. Most famously, weak, polyvalent interactions, often including RNA, may lead to a liquid-liquid phase separation. This effect greatly enhances local concentrations and is thought to promote interactions that would remain rare in dilute solution. Synthetic systems provide a means to clarify the underlying biophysical mechanisms at play, both in vitro and in the cell via exogenous expression. In this regard, ferritin is a useful substrate, as its composition of 24 subunits with octahedral symmetry supports self-assembly by close packing in 3D. The conventional diagnostic tool for protein condensation is fluorescence imaging. In this work we explore the use of refractive index mapping to detect states of condensation and decondensation. Using two related ferritin-based self-assembly systems, we find that refractive index is a sensitive indicator for reversible condensation. Surprisingly, refractive index indicates a rapid decondensation even when molecular dispersal kinetics are slow according to fluorescence. Conversely, in a photo-activated condensation where long activation results in slow decondensation kinetics, the refractive index provides reliable evidence for the physical state independent of fluorescence. The observations suggest a distinction between condensation to a sparse biomolecular network, or to a material continuum that supports an optical polarizability distinct from that of the dilute phase in solution.

biophysics↗

bioRxiv2024

Snapshots of Mitochondrial Fission Imaged by Cryo-Scanning Transmission Electron Tomography

Mitochondria undergo constant remodeling via fission, fusion, extension, and degradation. Fission, in particular, depends on the accumulation of the mitochondrial fission factor (MFF) and subsequent recruitment of the dynamin-related protein Drp1. We used cryo-scanning transmission electron tomography (cryo-STET) to investigate mitochondrial morphologies in MFF mutant cells (MFF-/-) in ATP-depleting conditions that normally induce fission. The capability of cryo-STET to image through the cytoplasmic volume to a depth of 1 {micro}m provides visualization of mitochondria and their surroundings intact. We imaged changes in mitochondrial morphology and cristae structure and contacts with endoplasmic reticulum, degradative organelles, and cytoskeleton at stalled fission sites. We found disruption of the outer membrane at contact sites with endoplasmic reticulum and degradative organelles at sites of mitophagy. We identified fission sites where the inner mitochondrial membrane is already separated while the outer is still continuous. While MFF is a general fission factor, these observations demonstrate that mitochondrial fission can proceed to the final stage in its absence. The use of cryo-STET allays concerns about the loss of structures due to sample thinning required for cryo-TEM tomography. Summary StatementImaging the whole cytosol in three dimensions greatly aids in understanding cellular processes. Here, we applied cryo-scanning transmission electron tomography to study stages of mitochondrial fission in the absence of the mitochondrial fusion factor protein.

cell biology↗

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↗

bioRxiv2022

Bridging the light-electron resolution gap with correlative cryo-SRRF and dual-axis cryo-STEM tomography

Cryo-electron tomography (cryo-ET) is the prime method for cell biological studies in three dimensions (3D) at high resolution. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access larger 3D volumes, on the scale of 1 micron, making it ideal to study organelles and their interactions in situ. Here we introduce two relevant advances: a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy (cryo-CLEM). Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to isotropic resolution in the reconstruction without averaging. We show individual protein densities in a mitochondrion-ER contact in a cell region 850 nm thick. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

cell biology↗

bioRxiv2022

3D electron diffraction reveals the chiral nature of the malaria pigment crystal hemozoin

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. X-ray powder diffraction has established that the unit cell contains a cyclic hematin dimer, yet the pro-chiral nature of heme supports formation of four distinct stereoisomers, two centrosymmetric and two chiral enantiomers. Here we apply emerging methods of in situ cryo-electron tomography and diffraction to obtain a definitive structure of biogenic hemozoin. Individual crystals take a striking polar morphology. Diffraction analysis, supported by density functional theory, indicates a compositional mixture of one centrosymmetric and one chiral dimer, whose absolute configuration has been determined on the basis of crystal morphology and interaction with the aqueous medium. Structural modeling of the heme detoxification protein suggests a mechanism for dimer selection. The refined structure of hemozoin should serve as a guide to new drug development.

microbiology↗

bioRxiv2022

Diffraction contrast in cryo-scanning transmission electron tomography reveals the boundary of hemozoin crystals in situ

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasites digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in vivo, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.

biophysics↗

bioRxiv2021

A Proposed Unified Mitotic Chromosome Architecture

A molecular architecture is proposed for an example mitotic chromosome, human Chromosome 10. This architecture is built on a previously described interphase chromosome structure based on Cryo-EM cellular tomography (1), thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle, for mitotic chromosomes, is specific coiling of the 11-nm nucleosome fiber into large scale approximately 200 nm structures (a Slinky (2, motif cited in 3) in interphase, and then further modification and subsequent additional coiling for the final structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well known cytological configurations. In addition, proof is experimentally provided, by digital PCR technology, that G1 T-cell nuclei are diploid, thus one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils. Significance StatementThe significance of this proposed mitotic chromosome architecture is that a specific, sequenced chromosome, human Chromosome 10, can be built into a specific architecture that accounts for the dimensional values and cytological descriptions, a first time result. Since this molecular architecture is an extension of the interphase chromosome structure, a coiling of the 11-nm nucleosome fiber with further coiling, a unifying molecular structure motif is present throughout the entire mitotic cycle, interphase through mitosis.

cell biology↗

bioRxiv2021

A Proposed Unified Interphase Nucleus Chromosome Structure: Preliminary Preponderance of Evidence

Cellular cryo-electron tomography (CET) of the cell nucleus using Scanning Transmission Electron Microscopy (STEM) and the use of deconvolution (DC) processing technology has highlighted a large-scale, 100-300 nm interphase chromosome structure (LSS), that is present throughout the nucleus. This chromosome structure appears to coil the nucleosome 11-nm fiber into a defined hollow structure, analogous to a Slinky (S) (1, motif used in 2) helical spring. This S architecture can be used to build chromosome territories, extended to polytene chromosome structure, as well as to the structure of Lampbrush chromosomes. Significance StatementCryo-preservation of the nuclear interior allows a large scale interphase chromosome structure--present throughout the nucleus--to be seen for the first time. This structure can be proposed to be a defined coiled entity, a Slinky. This structure can be further used to explain polytene chromosome structure, an unknown chromosome architecture as well as for lampbrush chromosomes. In addition, this new structure can be further organized as chromosome territories, using all 46 human interphase chromosomes as an example, easily into a 10 micron diameter nucleus. Thus, interphase chromosomes can be unified into a flexible defined structure.

cell biology↗

bioRxiv2021

Entropy Regularized Deconvolution of Cellular Cryo-Transmission Electron Tomograms

Cryo-electron tomography (cryo-ET) allows for the high resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy regularized deconvolution (ER DC) to cryo-electron tomography data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied DC to several in situ cryo-ET data sets, and assess the results by Fourier analysis and subtomogram analysis (STA).

biophysics↗

bioRxiv2020

3D Deconvolution Processing for STEM Cryo-Tomography

The complex environment of biological cells and tissues has motivated development of three dimensional imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryo-scanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles, and then in representative cryo-tomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin. Significance statementElectron tomography is used to reveal the structure of cells in three dimensions. The combination with cryogenic fixation provides a snapshot in time of the living state. However, cryo-tomography normally requires very thin specimens due to image formation by conventional phase contrast transmission electron microscopy (TEM). The thickness constraint can be relaxed considerably by scanning TEM (STEM), yet three-dimensional (3D) reconstruction is still subject to artifacts inherent in the collection of data by tilted projections. We show here that deconvolution algorithms developed for fluorescence microscopy can suppress these artifacts, resulting in significant contrast enhancement. The method is demonstrated by cellular tomography of complex membrane structures, and by segmentation of chromatin into distinct, contiguous domains of heterochromatin and euchromatin at high and low density, respectively.

biophysics↗