Foundational women in planetary geomorphology: Some contributions in fluvial, aeolian, and (cryo)volcanic subdisciplines
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Devon M. Burr↗
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Extracellular filaments revealed by affinity capture cryogenic-electron tomography
Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high-resolution imaging of other adherent and non-adherent cell types with cryo-ET.
Biochemistry↗
Cryogenic electron tomography by the numbers: Charting underexplored lineages in structural cell biology
Imaging cells and their interactions across the whole biosphere with molecular-scale resolution is key for understanding structure–function relations. Cryogenic electron tomography (cryo-ET) is a powerful method for obtaining this critical information. However, cryo-ET studies are challenging and often limited to a small number of cell types per study. Here, we collate cryo-ET data from hundreds of cells and tissues across the biosphere to i) identify emerging methodological trends, ii) pinpoint strategies to reduce imaging time and costs, iii) quantitatively compare methods for cell freezing and sectioning, and iv) census cryo-ET species coverage across all domains of life. Comparing the fraction of cellular material within a single lamella across all domains of life reveals an order of magnitude difference between eukaryotes (1%) compared to bacteria (9%) and archaea (14%). We calculate the fraction of cellular material which can be imaged using distinct sectioning methods on multicellular communities and tissues—identifying serial lift-out as a powerful approach for obtaining more complete cellular depictions. Finally, we show that the biodiversity of current cryo-ET studies is 2 to 3 orders of magnitude lower than in sequence libraries and 4 to 5 lower than the total predicted on Earth. Our analyses reveal major evolutionary lineages which remain critically understudied and highlight where future cryo-ET research would be most impactful.
HPF↗
Time-resolved cryogenic electron tomography for the study of transient cellular processes
Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second prior to freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.
59 BASIC BIOLOGICAL SCIENCES↗
Graph Identification of Proteins in Tomograms (GRIP-Tomo) 2.0: Topologically aware classification for proteins
Cryo-electron tomography (cryo-ET) enables structural characterization of biomolecules under near-native conditions. Existing approaches for interpreting the resulting three-dimensional volumes are computationally expensive and have difficulty interpreting density associated with small proteins/complexes. To explore alternate approaches for identifying proteins in cryo-ET data we pursued a Graph Network and topologically invariant approach. Here, we report on a fast algorithm that classifies particles by searching for nuances of evolutionarily conversed motifs and the geometrical characteristics of protein structure. GRIP-Tomo 2.0 is a machine-learning pipeline that extracts interpretable topological features of protein structures within noisy experimental backgrounds. Compared to version 1.0, the new pipeline includes three upgrades that significantly improve performance including synthetic tomogram generation simulating realistic noise, graph-based persistent feature extraction as protein fingerprints, and high-performance computing acceleration. GRIP-Tomo 2.0 achieves over 90% accuracy in classifying between proteins and noise using both real and synthetic datasets which represents a foundational step toward advancing cryo-ET workflows and empowering automated visual proteomics.
Li, Chengxuan↗
High-Resolution Photoelectron Spectroscopy of NO3 – Vibrationally Excited Along Its ν3 Mode
The nitrate (NO3) radical has long been the subject of both experimental and theoretical studies due to its complex electronic structure resulting from vibronic interactions between its X̃2A2' and B̃2E' states. In particular, the definite assignment of the fundamental of its degenerate stretching vibration (ν3) is still under debate. Here, we report high-resolution photoelectron spectra of vibrationally pre-excited NO3- using the recently developed IR-cryo-SEVI technique. The anions are excited through infrared (IR) excitation near 1350 cm-1, accessing the ν3 and 2ν3(e') vibrational levels with band centers at 1350.5 and ∼2700 cm-1, respectively. The IR-cryo-SEVI spectrum for 2ν3 pre-excitation shows clear evidence for an intense 321 transition. From the position of this feature (30031 cm-1), the electron affinity of NO3 also determined in this work (31680 cm-1), and the IR excitation energy, we obtain a fundamental frequency of 1051 cm-1 for the ν3 fundamental of the NO3 radical. This assignment and other features in the IR-cryo-SEVI spectra are supported by spectral simulations based on a vibronic Köppel-Domcke-Cederbaum Hamiltonian. The simulations also show that nearly all features in the IR-cryo-SEVI spectra arise because of pseudo-Jahn-Teller coupling between the X̃ and B̃ states of NO3. The results and analysis presented here settle a long-standing controversy regarding the ν3 frequency of NO3.
Anions↗
De novo atomic protein structure modeling for cryoEM density maps using 3D transformer and HMM
Accurately building 3D atomic structures from cryo-EM density maps is a crucial step in cryo-EM-based protein structure determination. Converting density maps into 3D atomic structures for proteins lacking accurate homologous or predicted structures as templates remains a significant challenge. Here, we introduce Cryo2Struct, a fully automated de novo cryo-EM structure modeling method. Cryo2Struct utilizes a 3D transformer to identify atoms and amino acid types in cryo-EM density maps, followed by an innovative Hidden Markov Model (HMM) to connect predicted atoms and build protein backbone structures. Cryo2Struct produces substantially more accurate and complete protein structural models than the widely used ab initio method Phenix. Additionally, its performance in building atomic structural models is robust against changes in the resolution of density maps and the size of protein structures.
59 BASIC BIOLOGICAL SCIENCES↗
Optical interference for the guidance of cryogenic focused ion beam milling beyond the axial diffraction limit
Cryogenic focused ion beam (Cryo-FIB) milling has become a standard step in the cryogenic electron tomography (Cryo-ET) workflow and is required to thin cells to electron-semitransparency. However, this destructive process removes the vast majority of the cellular material and raises a critical question: what thin section should be preserved for Cryo-ET analysis? Using a tri-coincident cryogenic FIB-SEM-LM system, we identify an interferometric optical response that can be used for targeting lamella production to fluorescently labeled structures with accuracy beyond the diffraction limit. Here we demonstrate this approach using synthetic samples of fluorescent beads embedded in micron-scale droplets of amorphous ice. We then apply the approach to capture virions inside host cells. Successful targeting is confirmed by Cryo-ET revealing clusters of virions in intracellular vesicles. The method does not require any fluorescent fiducials or axial registration and can be performed on any fluorescently labeled structure that is visible in widefield fluorescence microscopy.
Cryoelectron microscopy↗
Complex water networks visualized by cryogenic electron microscopy of RNA
The stability and function of biomolecules are directly influenced by their myriad interactions with water. Here we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule: the Tetrahymena ribozyme. By using segmentation-guided water and ion modelling (SWIM), an approach combining resolvability and chemical parameters, we automatically modelled and cross-validated water molecules and Mg 2+ ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics, supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modelling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and molecular dynamics simulations.
59 BASIC BIOLOGICAL SCIENCES↗
Operando Freezing Cryogenic Electron Microscopy of Active Battery Materials
Abstract Understanding structural and chemical evolution of battery materials during operation is critical to achieving safe, efficient, and long-lasting energy storage. Cryogenic electron microscopy (cryo-EM) has become a valuable tool in battery characterization, leveraging low temperatures to improve stability of sensitive materials under electron beam irradiation. However, typical cryo-EM sample preparations leave extended time between the electrochemical point of interest and ex situ freezing of samples, during which active structures may relax, degrade, or otherwise evolve. Here, we detail a method for operando freezing cryo-EM to preserve and characterize native electrode and interfacial structures that arise during battery cycling, based on an operando plunge freezer and cold sample removal process. We validate the method on multiple electrode materials and quantify and discuss the freezing rate achieved. Operando freezing cryo-EM can be used to directly visualize transient features that arise at active electrochemical interfaces, to enable deeper understanding of structural evolution and interfacial chemistry in batteries and other electrochemical systems.
25 ENERGY STORAGE↗
Human NLRP3 inflammasome activation leads to formation of condensate at the microtubule organizing center
The NLRP3 inflammasome is a multiprotein molecular machine that drives inflammatory responses in innate immunity. Although its dysregulation is implicated in numerous human diseases, its structural organization in cells remains poorly understood. Here, we used precise fluorescence-guided cryo–focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to visualize NLRP3 inflammasomes in situ within human macrophages at various stages of activation. After priming and activation, we observed expansion and dispersion of Golgi cisternae, along with the emergence of 50-nanometer NLRP3-associated vesicles, which likely transport NLRP3 to the MTOC. Dense NLRP3-containing condensates then formed in and around the MTOC. In later stages, the condensates solidified, coincident with widespread mitochondrial damage, autophagy, and pyroptotic cell death.
Wang, Jue [Division of Chemistry and Chemical Engi↗
Alignment and evaluation of the cryogenic corrected infrared astronomical satellite /IRAS/ telescope
Room temperature alignment and evaluation techniques for the Infrared Astronomical Satellite (IRAS) telescope, which has a primary mirror figured to correct for surface distortions and the 2 K operating temperature are discussed. Interferometric cryogenic testing of the 0.6 m, f/1.5 lightweighted beryllium primary mirror at its intended operating temperature reveals surface distortions that can be modeled with Zernike polynomials. With this model, it becomes possible to derive the 'inverse' of the cryowavefront error (ideal cryo mirror) and to figure the cryo correction into the primary mirror using Perkin-Elmer's Computer Controlled Polisher. It is recognized that during room temperature assembly of the system, misalignment of the secondary mirror can introduce additional unwanted aberrations that may cancel or distort the wavefront errors purposely introduced by the cryo figuring. To avoid this possible degradation and to ensure optimum telescope performance, the system Zernike polynomial coefficients and wavefront maps generated from the in-process alignment interferograms are monitored and compared to Zernike coefficients and wavefront maps for the cryo corrected primary mirror.
Harned, N.↗
Cryogenic Propellant Long-term Storage With Zero Boil-off
Significant boil-off losses of cryogenic propellant storage systems in long-duration space mission applications result in additional propellant and large tanks. The zero boil-off (ZBO) concept consists of an active cryo-cooling system integrated with traditional passive thermal insulation. The potential mass reductions with the ZBO concept are Substantial; therefore, further exploration through technology programs has been initiated within NASA. A large-scale demonstration of the ZBO concept has been devised utilizing the Marshall Space Flight Center (MSFC) Multipurpose Hydrogen Test Bed (MHTB) along with a cryo-cooler unit. The cryo-cooler with the MHTB and spraybar recirculation/mixer system in a manner that enables thermal energy removal at a rate that equals the total tank heat leak. The liquid hydrogen is withdrawn from the tank, passed through a heat exchanger, and then the chilled liquid is sprayed back into the tank through a spraybar. The test series will be performed over a 30-40 day period. Tests will be conducted at multiple fill levels and various mixer operational cycles to demonstrate concept viability and to provide benchmark data to be used in analytical model development. In this paper. analytical models for heat flows through the MHTB tank, cryo-cooler performance. and spraybar performance will be presented.
Hedayat, A.↗
Perfectly Cold Crystals: What Happens When They Are X-rayed?
For many macromolecular crystals the cryo-preservation of these crystals during X-ray data collection is of crucial importance, particularly at synchrotron facilities where the crystals rapidly receive a high dose of radiation. A practical variable to ensure adequate preservation is the variation of the cryo-protectant present when the crystal is preserved. Our initial approach to study X-ray diffraction data quality as a function of cryo-protectant present when preserving a xylose isomerase crystal shows that the data quality can be tremendously improved by recipe adjustment. Guided by crystal mosaicity estimates, we optimized crystal growth conditions to obtain cryo-preserved xylose isomerase crystals that withstand a very high dose of X-rays, with only the smallest amount of radiation damage at ultra-high resolution (1.2Angstroms). The rate at which damage occurs allowed us to collect a series of complete data sets, which show how the data degradation proceeds over time. We are here presenting data for the xylose isomerase crystallization recipe improvement and our interpretation of the crystal degradation process during X-ray data collection.
vanderWoerd, Mark↗
Continuously Regenerable Freeze-Out CO2 Control Technology
Carbon dioxide (CO2) removal technology development for portable life support systems (PLSS) has traditionally concentrated in the areas of solid and liquid chemical sorbents and semi-permeable membranes. Most of these systems are too heavy in gravity environments, require prohibitive amounts of consumables for operation on long term planetary missions, or are inoperable on the surface of Mars due to the presence of a CO2 atmosphere. This paper describes the effort performed to mature an innovative CO2 removal technology that meets NASA s planetary mission needs while adhering to the important guiding principles of simplicity, reliability, and operability. A breadboard cryogenic carbon dioxide scrubber (Cryo Scrubber) for a closed loop cryogenic PLSS was developed, designed, and tested, and a conceptual design suitable for a PLSS was developed based on the results of the breadboard testing. The Cryo Scrubber freezes CO2 and other trace contaminants out of expired vent loop gas using cooling available from a liquid oxygen (LOX) based PLSS. The device is continuously regenerable, with solid CO2 being removed from the cold freeze-out surfaces, sublimated, and vented overboard. Duration is limited only by the supply of LOX stored in the PLSS. Simplicity, reliability, and operability are universally important criteria for critical hardware on long duration Lunar or Mars missions. The Cryo Scrubber has no moving parts, requires no additional consumables, and uses no electrical power, contributing to its simplicity and reliability. It is easy to use; no operator action is required to prepare, use, or shut down the Cryo Scrubber, and it does not require charging or regeneration. The versatility of the concept allows for operation on earth, the moon, and Mars, and in microgravity.
Fricker, John↗
Preliminary experimental studies into the storage capacity of cryogenic hydrogen in aerogel blanket materials
The abundance and diversity of hydrogen applications necessitates continued and accelerated research into advanced storage technologies. Traditionally, hydrogen has been stored as either a high-pressure, warm gas; or a low-pressure, cryogenic liquid. Methods such as cryo-supercritical and cryo-adsorbed have been explored, but are not yet mainstream. Cryo-adsorbed is attractive because higher storage densities at higher temperatures than liquid may be achieved. Recently NASA, in partnership with Eta Space, Southwest Research Institute, the University of Central Florida, and Air Liquide, have been exploring the use of inexpensive, commercially available silica aerogel blanket materials for cryo-adsorbed hydrogen storage. Unlike most adsorbents, aerogel blanket is not a powder, but a robust, composite material that can be formed into complex shapes to aid in more efficient storage system designs, and has already been proven to uptake large quantities of fluids such as nitrogen and oxygen. Recent experimental efforts into the uptake of low-pressure hydrogen gas at 77 K, and liquid hydrogen at normal boiling point (NBP) will be discussed. Although preliminary in nature, the test results are promising, showing up to a 49% increase in storage density at 77 K over the gas alone, and greater than a one-to-one volume equivalency with NBP LH 2 .
A M Swanger↗
Fundamental Understanding of “Fresh” Lithium Nucleation and Growth in Sulfide-Based Anode-Free Solid-State Batteries: Effects of Substrate, Current Density, and Li + Supply
Understanding the electrochemical extraction and deposition of lithium (Li) from cathode is crucial for advancing anode-free solid-state batteries (AFSSBs). Herein, cryo-transmission electron microscopy (cryo-TEM) and electrochemical studies are employed to investigate how current collector surface properties, current densities, and cathode loadings influence the morphology of fresh electrochemically deposited Li and the electrochemical performance in sulfide-based AFSSBs. Cryo-TEM reveals that Cu current collectors induce irregular, dendritic Li deposits due to their lithiophobic nature and reactivity with Li 5.4 PS 4.4 Cl 1.6 (LPSC), while Ni and Au facilitate more uniform, planar-like Li growth. The morphology of the deposited Li also depends on current density: higher rates produce smaller, porous particles versus larger, denser deposits at lower rates. Importantly, for the first time, we discovered that low cathode loadings lead to poor cycling stability due to insufficient Li + supply for complete anode coverage, contrary to conventional solid-state batteries with preloaded Li metal anodes. This finding establishes a design principle where adequate cathode Li + reservoirs are essential for anode interface stability in AFSSBs. Overall, this work elucidates the interplayed effect of current collectors, current densities, and cathode loadings on Li morphology and cycle stability, offering fundamental insights into cathode-derived Li behavior and practical guidelines for optimizing AFSSBs performance through nucleation control and interface engineering.
25 ENERGY STORAGE↗
Structural Complexities in Sodium Ion Conductive Antiperovskite Revealed by Cryogenic Transmission Electron Microscopy
Here we use low-dose cryogenic transmission electron microscopy (cryo-TEM) to investigate the atomic-scale structure of antiperovskite Na 2 NH 2 BH 4 crystals by preserving the room-temperature cubic phase and carefully monitoring the electron dose. Via quantitative analysis of electron beam damage using selected area electron diffraction, we find cryogenic imaging provides 6-fold improvement in beam stability for this solid electrolyte. Cryo-TEM images obtained from flat crystals revealed the presence of a new, long-range-ordered supercell with a cubic phase. The supercell exhibits doubled unit cell dimensions of 9.4 Å × 9.4 Å as compared to the cubic lattice structure revealed by X-ray crystallography of 4.7 Å × 4.7 Å. The comparison between the experimental image and simulated potential map indicates the origin of the supercell is a vacancy ordering of sodium atoms. This work demonstrates the potential of using cryo-TEM imaging to study the atomic-scale structure of air- and electron-beam-sensitive antiperovskite-type solid electrolytes.
36 MATERIALS SCIENCE↗