scholarly journals Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Mohamed Koronfel ◽  
Ilias Kounatidis ◽  
Dennis M. Mwangangi ◽  
Nina Vyas ◽  
Chidinma Okolo ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Super Resolution ◽  
X Rays ◽  
Imaging Data ◽  
Filamentous Actin ◽  
Intracellular Space ◽  
X Ray ◽  
Integral Role

Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.

Relative Survival of Hybrid X-Ray-Resistant, and Normally Sensitive Mammalian Cells Exposed to X Rays and Protons under Aerobic and Hypoxic Conditions

1978 ◽  
Vol 73 (3) ◽  
pp. 585 ◽  
Author(s):  
Jerry R. Williams ◽  
Robert G. Gould ◽  
Daniel Flynn ◽  
James B. Robertson ◽  
John B. Little
Keyword(s):  
Mammalian Cells ◽  
Relative Survival ◽  
X Rays ◽  
X Ray ◽  
Hypoxic Conditions

scholarly journals Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source

2014 ◽  
Vol 21 (6) ◽  
pp. 1370-1377 ◽  
Author(s):  
Mark A. Le Gros ◽  
Gerry McDermott ◽  
Bertrand P. Cinquin ◽  
Elizabeth A. Smith ◽  
Myan Do ◽  
...  
Keyword(s):  
Data Collection ◽  
Light Source ◽  
Mammalian Cells ◽  
National Laboratory ◽  
Three Dimensional ◽  
Imaging Method ◽  
X Rays ◽  
X Ray ◽  
Correlative Imaging ◽  
Advanced Light Source

Beamline 2.1 (XM-2) is a transmission soft X-ray microscope in sector 2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. XM-2 was designed, built and is now operated by the National Center for X-ray Tomography as a National Institutes of Health Biomedical Technology Research Resource. XM-2 is equipped with a cryogenic rotation stage to enable tomographic data collection from cryo-preserved cells, including large mammalian cells. During data collection the specimen is illuminated with `water window' X-rays (284–543 eV). Illuminating photons are attenuated an order of magnitude more strongly by biomolecules than by water. Consequently, differences in molecular composition generate quantitative contrast in images of the specimen. Soft X-ray tomography is an information-rich three-dimensional imaging method that can be applied either as a standalone technique or as a component modality in correlative imaging studies.

scholarly journals Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers

2020 ◽  
Vol 117 (11) ◽  
pp. 5861-5872 ◽  
Author(s):  
Carsten F. E. Schroer ◽  
Lucia Baldauf ◽  
Lennard van Buren ◽  
Tsjerk A. Wassenaar ◽  
Manuel N. Melo ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Mechanical Stability ◽  
Coarse Grained ◽  
Lipid Bilayer Membranes ◽  
Filamentous Actin ◽  
Divalent Ions

The cytoskeletal protein actin polymerizes into filaments that are essential for the mechanical stability of mammalian cells. In vitro experiments showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biological design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained molecular dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly determine the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated experimentally by reconstitution assays with actin on lipid bilayer membranes and provide a molecular-level understanding of the actin–membrane interaction.

scholarly journals Long-Range and Directional Allostery of Actin Filaments Plays Important Roles in Various Cellular Activities

2020 ◽  
Vol 21 (9) ◽  
pp. 3209 ◽  
Author(s):  
Kiyotaka Tokuraku ◽  
Masahiro Kuragano ◽  
Taro Q. P. Uyeda
Keyword(s):  
Long Range ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Intracellular Transport ◽  
Structural Changes ◽  
Scale Space ◽  
Actin Binding ◽  
Cell Interior ◽  
Intracellular Space ◽  
Micrometer Scale

A wide variety of uniquely localized actin-binding proteins (ABPs) are involved in various cellular activities, such as cytokinesis, migration, adhesion, morphogenesis, and intracellular transport. In a micrometer-scale space such as the inside of cells, protein molecules diffuse throughout the cell interior within seconds. In this condition, how can ABPs selectively bind to particular actin filaments when there is an abundance of actin filaments in the cytoplasm? In recent years, several ABPs have been reported to induce cooperative conformational changes to actin filaments allowing structural changes to propagate along the filament cables uni- or bidirectionally, thereby regulating the subsequent binding of ABPs. Such propagation of ABP-induced cooperative conformational changes in actin filaments may be advantageous for the elaborate regulation of cellular activities driven by actin-based machineries in the intracellular space, which is dominated by diffusion. In this review, we focus on long-range allosteric regulation driven by cooperative conformational changes of actin filaments that are evoked by binding of ABPs, and discuss roles of allostery of actin filaments in narrow intracellular spaces.

scholarly journals Coronal Transients in Radio and X-rays

1996 ◽  
Vol 154 ◽  
pp. 15-22
Author(s):  
Mukul R. Kundu
Keyword(s):  
Active Region ◽  
Spatial Resolution ◽  
High Spatial Resolution ◽  
X Rays ◽  
Imaging Data ◽  
X Ray ◽  
Type Iii ◽  
Wave Data ◽  
Spatially Resolved ◽  
Radio Imaging

AbstractWe present a summary of several studies of transient coronal phenomena based upon high spatial resolution radio imaging data along with Yohkoh SXT and HXT observations. In addition to normal flares the studies also involve such exotic events as active region transient brightenings (ARTB) and coronal jets and bright points. We provide evidence of nonthermal processes in flaring X-ray bright points from spatially resolved meter-wave data, existence and propagation of type III burst emitting electrons in coronal jets, radio signatures of ARTB’s, and beaming of electrons producing microwave and hard X-rays. The implications of these observations are discussed.

scholarly journals A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

1994 ◽  
Vol 14 (7) ◽  
pp. 4741-4748 ◽  
Author(s):  
W K Rathmell ◽  
G Chu
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
X Rays ◽  
X Ray ◽  
Break Repair ◽  
Group 5 ◽  
Dna Ends

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

scholarly journals FLANNEL  (Focal Loss bAsed Neural Network EnsembLe) for COVID-19 detection

2020 ◽  
Author(s):  
Zhi Qiao ◽  
Austin Bae ◽  
Lucas M Glass ◽  
Cao Xiao ◽  
Jimeng Sun
Keyword(s):  
Neural Network ◽  
Convolutional Neural Network ◽  
Class Imbalance ◽  
X Rays ◽  
Neural Network Ensemble ◽  
Imaging Data ◽  
Identification Task ◽  
Neural Network Models ◽  
X Ray ◽  
Chest X Ray

Abstract Objective The study sought to test the possibility of differentiating chest x-ray images of coronavirus disease 2019 (COVID-19) against other pneumonia and healthy patients using deep neural networks. Materials and Methods We construct the radiography (x-ray) imaging data from 2 publicly available sources, which include 5508 chest x-ray images across 2874 patients with 4 classes: normal, bacterial pneumonia, non–COVID-19 viral pneumonia, and COVID-19. To identify COVID-19, we propose a FLANNEL (Focal Loss bAsed Neural Network EnsembLe) model, a flexible module to ensemble several convolutional neural network models and fuse with a focal loss for accurate COVID-19 detection on class imbalance data. Results FLANNEL consistently outperforms baseline models on COVID-19 identification task in all metrics. Compared with the best baseline, FLANNEL shows a higher macro-F1 score, with 6% relative increase on the COVID-19 identification task, in which it achieves precision of 0.7833 ± 0.07, recall of 0.8609 ± 0.03, and F1 score of 0.8168 ± 0.03. Discussion Ensemble learning that combines multiple independent basis classifiers can increase the robustness and accuracy. We propose a neural weighing module to learn the importance weight for each base model and combine them via weighted ensemble to get the final classification results. In order to handle the class imbalance challenge, we adapt focal loss to our multiple classification task as the loss function. Conclusion FLANNEL effectively combines state-of-the-art convolutional neural network classification models and tackles class imbalance with focal loss to achieve better performance on COVID-19 detection from x-rays.

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination

1994 ◽  
Vol 14 (7) ◽  
pp. 4741-4748
Author(s):  
W K Rathmell ◽  
G Chu
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
X Rays ◽  
X Ray ◽  
Break Repair ◽  
Group 5 ◽  
Dna Ends

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

scholarly journals Staircase array of inclined refractive multi-lenses for large field of view pixel super-resolution scanning transmission hard X-ray microscopy

2021 ◽  
Vol 28 (3) ◽  
Author(s):  
Talgat Mamyrbayev ◽  
Katsumasa Ikematsu ◽  
Hidekazu Takano ◽  
Yanlin Wu ◽  
Kenji Kimura ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Super Resolution ◽  
High Energy ◽  
Two Dimensions ◽  
Field Of View ◽  
Large Field ◽  
Acquisition Time ◽  
X Rays ◽  
X Ray ◽  
Scanning Transmission

Owing to the development of X-ray focusing optics during the past decades, synchrotron-based X-ray microscopy techniques allow the study of specimens with unprecedented spatial resolution, down to 10 nm, using soft and medium X-ray photon energies, though at the expense of the field of view (FOV). One of the approaches to increase the FOV to square millimetres is raster-scanning of the specimen using a single nanoprobe; however, this results in a long data acquisition time. This work employs an array of inclined biconcave parabolic refractive multi-lenses (RMLs), fabricated by deep X-ray lithography and electroplating to generate a large number of long X-ray foci. Since the FOV is limited by the pattern height if a single RML is used by impinging X-rays parallel to the substrate, many RMLs at regular intervals in the orthogonal direction were fabricated by tilted exposure. By inclining the substrate correspondingly to the tilted exposure, 378000 X-ray line foci were generated with a length in the centimetre range and constant intervals in the sub-micrometre range. The capability of this new X-ray focusing device was first confirmed using ray-tracing simulations and then using synchrotron radiation at BL20B2 of SPring-8, Japan. Taking account of the fact that the refractive lens is effective for focusing high-energy X-rays, the experiment was performed with 35 keV X-rays. Next, by scanning a specimen through the line foci, this device was used to perform large FOV pixel super-resolution scanning transmission hard X-ray microscopy (PSR-STHXM) with a 780 ± 40 nm spatial resolution within an FOV of 1.64 cm × 1.64 cm (limited by the detector area) and a total scanning time of 4 min. Biomedical implant abutments fabricated via selective laser melting using Ti–6Al–4V medical alloy were measured by PSR-STHXM, suggesting its unique potential for studying extended and thick specimens. Although the super-resolution function was realized in one dimension in this study, it can be expanded to two dimensions by aligning a pair of presented devices orthogonally.

White-Light Coronal Structures: Streamers and CMEs

1994 ◽  
Vol 144 ◽  
pp. 82
Author(s):  
E. Hildner
Keyword(s):  
Emission Line ◽  
Electron Density ◽  
White Light ◽  
Coronal Mass Ejections ◽  
X Rays ◽  
Solar Cycles ◽  
Temperature Function ◽  
X Ray ◽  
Eclipse Observations ◽  
Coronal Structures

AbstractOver the last twenty years, orbiting coronagraphs have vastly increased the amount of observational material for the whitelight corona. Spanning almost two solar cycles, and augmented by ground-based K-coronameter, emission-line, and eclipse observations, these data allow us to assess,inter alia: the typical and atypical behavior of the corona; how the corona evolves on time scales from minutes to a decade; and (in some respects) the relation between photospheric, coronal, and interplanetary features. This talk will review recent results on these three topics. A remark or two will attempt to relate the whitelight corona between 1.5 and 6 R⊙to the corona seen at lower altitudes in soft X-rays (e.g., with Yohkoh). The whitelight emission depends only on integrated electron density independent of temperature, whereas the soft X-ray emission depends upon the integral of electron density squared times a temperature function. The properties of coronal mass ejections (CMEs) will be reviewed briefly and their relationships to other solar and interplanetary phenomena will be noted.

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