BER Structural Biology and Imaging Resources
Synchrotron, Neutron, and Cryo-EM
U.S. Department of Energy | Office of Science | Office of Biological and Environmental Research

Soft X-ray Tomography

High-resolution 3D cellular imaging

E. coli Cell Nucleoid Organization. Soft X-ray tomographic (SXT) imaging shows the organization of cell nucleoids (yellow) within cells (clear or blue background) of two different strains of E. coli (top, wild type strain MG1655; bottom, Hua38 mutant strain SK3842). Various cell morphologies found to be representative of the three growth stages are shown at left (lag phase), middle (exponential phase), and right (stationary phase). [From Hammel, M. H., et al. 2016.]

Soft X-ray tomography (SXT) is a non-invasive, 3D imaging technique that can measure volumes, surfaces, interfaces, membranes, and organelle connectivity within intact cells. SXT data are collected on a transmission soft x-ray microscope, called XM-2, located at the Advanced Light Source. In SXT, the specimen is illuminated with photons at energies from within the “water window,” a region of the electromagnetic spectrum in which water is transparent to soft x-rays (i.e., between the K-absorption edge of carbon at 282 eV and oxygen at 533 eV). As the illumination passes through the specimen, it is attenuated according to the type and concentration of molecules in the specimen. Photons within the water window are more strongly absorbed by carbon-containing and nitrogen-containing organic materials than they are by water by a full order of magnitude. Consequently, subtle differences in biochemical composition produce measurable contrast in soft x-ray images. SXT data currently have spatial resolutions of 35 or 50 nm, depending on the type of information required and the type of specimen being imaged. Consequently, SXT visualizes structures ranging from molecular machines to entire cells.

Key Features of Soft X-ray Tomography

  • High spatial resolution: full-field imaging is possible at spatial resolutions of 35 nm or better.
  • Broad applicability: microbes, yeast, and eukaryotes can be imaged intact and fully hydrated.
  • Quantitative image contrast mechanism: each voxel in a calculated image reconstruction has an associated linear absorption coefficient (LAC), a measurement which enables better visualization of cellular features.
  • High data completeness: specimens mounted in thin-walled glass capillaries can be imaged from any position around a central axis of rotation. This results in high-completeness tomographic data with no “missing wedges,” as is the case with image series using flat specimen holders with limited tilt. Specimens may also be mounted on flat substrates, as for electron microscopy, if necessary.
  • High specimen throughput: the acquisition of a full tomographic series (180 projection images), image alignment, and tomographic reconstruction of a field of view can take less than ten minutes. A subsequent field of view can be imaged by merely translating the specimen-containing capillary in the Z direction. A field of view can contain tens or even hundreds of bacteria, five to ten yeast cells, or one to three mammalian cells.

Algal Cell Morphology. Soft X-ray tomography provides a reconstructed cell with segmented nucleus (purple), chloroplast (green), mitochondria (red), lipids (yellow), and starch granules within the chloroplast (blue). (A) A representative orthoslice of the reconstructed cell. (B) Three-dimensional view. (C) Chloroplast and nucleus. (D) Fully segmented cell. [From Roth, M. S., et al. 2017.]

BER Researchers Use Soft X-ray Tomography to:

  • Image the subcellular organization of bacteria, yeast, and higher-order cells
  • Quantify biofuel production
  • Visualize and quantify the effects of exposure to environmental contaminants on cells
  • Study the influence of genetic factors on cell phenotype

See more examples in Science Highlights

Sample Considerations

  • Cells are cryo-fixed and imaged at atmospheric pressure. No staining or sectioning is required.
  • Biological specimens up to 15 µm thick and greater than 50 µm long are suitable.
  • The particulars of your specimen and data requirements should be discussed with beamline staff.

Soft X-ray Beamlines at DOE User Facilities

Each beamline has unique characteristics. To determine the user facility and beamline best suited to your science questions, see additional information and beamline contacts at the link below.

Citations

Hammel, M. H., et al. 2016. “HU multimerization shift controls nucleoid compaction,” Science Advances 2(7), e1600650. DOI: 10.1126/sciadv.1600650.

Roth, M. S., et al. 2017. “Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production,” PNAS 114(21), E4296-4305. DOI: 10.1073/pnas.1619928114.

Research Highlights

DateTitleTechniqueFacilityScience Topic
02/11/2021A Modular Approach to Designing Protein AssembliesSolution X-ray ScatteringAdvanced Light SourceMolecular Structure
01/19/2021Fusion Enzymes Improve Yield of Functionalized TerpenesSolution X-ray ScatteringAdvanced Light SourceMolecular Structure
01/06/2021Designing a Controllable Self-Assembling BiomaterialSolution X-ray ScatteringAdvanced Light SourceMolecular Structure
02/03/2020Antibodies Elicited by Zika Vaccine CharacterizedX-ray Macromolecular CrystallographyAdvanced Photon Source, National Synchrotron Light Source IIMolecular Structure
09/24/2019Revealing a MERS-CoV VulnerabilityX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
10/27/2020Potent Antibody Against SARS-CoV-2X-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
02/02/2021SARS-CoV-2 Enzyme Structure Reveals Potential InhibitorsX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
05/21/2021Essential Enzyme in Bacterial Protein SynthesisX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
06/02/2021Complexation of Metals with Bacterial MethanobactinsX-ray Absorption and Emission SpectroscopyStanford Synchrotron Radiation LightsourceChemical and Elemental Information
03/24/2021Deciphering the Mechanism of Enzymatic Methane SynthesisX-ray Absorption and Emission Spectroscopy, X-ray Fluorescence ImagingStanford Synchrotron Radiation LightsourceChemical and Elemental Information
10/30/2020Molecular Crystals Hybridized with Hydrogel Polymers Achieve Unprecedented Material Attributes
01/07/2020Multistep Crystallization of Microbial S-Layers
03/23/2020Antibiotics with a Built-in OFF Switch
11/02/2020Mechanistic Insights into Cu(I) Biogenesis of Galactose Oxidase
09/01/2020Tracking Foliar Zinc Absorption in Apple Trees
01/01/2021Soil Amendments and Flooding Cycle Impact Cadmium Bioavailability in Paddy Soil
08/18/2020Visualizing Enzymes in Action
01/22/2020Efficient and Economical Strategy for Functional Metalloprotein Design
03/10/2020Introducing Positive Charges in an Artificial Enzyme Increases CO2 Hydrogenation
12/09/2019Tuning the Catalytic Bias of Hydrogenase Enzymes
03/08/2018Safety Concerns Over Tungsten Accumulation in Bone
07/08/2019Nanoparticle Properties Influence Transport and Distribution in Plants
10/17/20183.7-Billion-Year-Old Rock Structures Formed by Tectonics, Not Life
09/07/2018First X-ray Nanofluorescence Tomography of Single Bacterial Cell
12/10/2018Nanoparticle Surface Chemistry Influences Journey Through Tomato Plant
11/07/2017Characterization of Elusive Yeast Protein Complex Involved in Autophagy
03/24/2020Critical Structural Differences Between Seasonal and Pandemic Influenza
03/24/2020Tunable Molecular Coating Stabilizes "DNA Origami" Structures
05/16/2019Key Drug Target Shown Assembling in Real Time
06/17/2021Surface Features of Prions Reveal Mechanism of Replication
01/10/2018Novel Architecture of a Bacterial Plasma Membrane Transporter
11/16/20173D Protein Structure Aids Search for Vaccine
08/09/2018Activation Mechanism of a Protein Kinase Reveals Role in Cancer Proliferation
10/08/2018Searching for New Ways to Fight Bacterial Resistance
06/21/2019Light Teaches Coenzymes New TricksX-ray Macromolecular CrystallographyNational Synchrotron Light Source IIMolecular Structure
05/21/2019Structural Basis of Bacterial Spore GerminationX-ray Macromolecular CrystallographyNational Synchrotron Light Source IIMolecular Structure
05/07/2020Mechanism of Activation of a Plant MetacaspaseX-ray Macromolecular CrystallographyNational Synchrotron Light Source IIMolecular Structure
04/14/2021Molecular Fragment Screening Identifies Potential SARS-CoV-2 InhibitorsX-ray Macromolecular CrystallographyAdvanced Light Source, National Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceMolecular Structure
06/13/2018Structure of a Nuclear Membrane GatekeeperX-ray Macromolecular CrystallographyNational Synchrotron Light Source IIMolecular Structure
09/23/2019Mechanism of Regulatory Protein's Dual Role as Inhibitor and ActivatorX-ray Macromolecular CrystallographyNational Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceMolecular Structure
04/02/2013Major Scaffold Component in Nuclear Pore ComplexSolution X-ray ScatteringStanford Synchrotron Radiation LightsourceMolecular Structure
05/24/2012Modulation of Plant Hormones by GH3 ProteinsX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
10/29/2013Common Physical Processes in Different Biomass PretreatmentsSmall-Angle Neutron Scattering, X-ray Macromolecular CrystallographySpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
07/17/2013Lessening X-ray Damage Speeds Protein DiscoveryX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
02/07/2013Lipid Bilayer Composition Regulates Function of Lipid Rafts in Model Cell MembranesSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
12/09/2012Watching the Evolution of a Protein’s FunctionX-ray Absorption and Emission SpectroscopyStanford Synchrotron Radiation LightsourceChemical and Elemental Information
08/15/2013Multifunctionality of an Ebola Virus ProteinX-ray Macromolecular CrystallographyAdvanced Light Source, Advanced Photon Source, Stanford Synchrotron Radiation LightsourceMolecular Structure
09/26/2014Understanding Nitrogen Fixation in BacteriaX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
11/10/2014Self-Assembling Porous Protein Cage Designed for Potential Biotech ApplicationsSolution X-ray Scattering, X-ray Macromolecular CrystallographyAdvanced Light SourceMolecular Structure
08/25/2015Modular Construction on a Biomolecular ScaleX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
06/10/2015Archaeal Enzyme Plays Key Role in Atmospheric Carbon TurnoverX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
09/08/2015Manganese Reduction-Oxidation Drives Plant Debris DecompositionSynchrotron Infrared Hyperspectral Imaging, X-ray Absorption and Emission Spectroscopy, X-ray Fluorescence ImagingAdvanced Light Source, Stanford Synchrotron Radiation LightsourceChemical and Elemental Information
12/07/2016Using Soft X-ray Ptychography to Investigate a Bacterium’s Inner CompassX-ray Absorption and Emission SpectroscopyAdvanced Light SourceChemical and Elemental Information
11/30/2016Crystal Structure of NOV1 Enzyme Reveals Mechanism of ActionX-ray Macromolecular CrystallographyAdvanced Light SourceMolecular Structure
11/15/2016Mapping the Migration of Genetic MaterialSoft X-ray TomographyAdvanced Light SourceCell and Tissue Structure
09/30/2020Understanding an Essential Chaperone ComplexCryo-Electron Microscopy, X-ray Macromolecular CrystallographyAdvanced Photon Source, National Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceMolecular Structure
07/31/2020Targeting a Critical Molecular Switch in COVID-19Solution X-ray ScatteringNational Synchrotron Light Source IIMolecular Structure
11/30/2020Fine Tuning Drugs to Fight CancerX-ray Macromolecular CrystallographyAdvanced Photon Source, National Synchrotron Light Source IIMolecular Structure
01/12/2016Cellulose Synthesis ComplexSmall-Angle Neutron Scattering, Solution X-ray ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
12/14/2016Hollow Pyramid Unlocks Principles of Protein ArchitectureSolution X-ray ScatteringAdvanced Light SourceMolecular Structure
12/20/2016Detailing the Molecular Roots of Alzheimer’s DiseaseX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
08/15/2016How Your Body Transports Zinc to Protect Your HealthX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
07/20/2017The CRISPR Target-Recognition MechanismX-ray Macromolecular CrystallographyAdvanced Light Source, Stanford Synchrotron Radiation LightsourceMolecular Structure
05/03/2018Dynamic Regulation of Histone Chaperone NucleoplasminSolution X-ray ScatteringStanford Synchrotron Radiation LightsourceMolecular Structure
06/23/2017Revealing How Bacterial Organelles AssembleX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
06/26/2017Molecular Basis of Alternative Tyrosine Biosynthesis in PlantsX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
04/12/2017Green Alga Genome Provides Blueprint to Advance Clean Energy, BioproductsSoft X-ray TomographyAdvanced Light SourceCell and Tissue Structure
10/06/2016Description of Hydration Water in Green Fluorescent Protein SolutionSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
09/29/2017Precise Control of Neutron Contrast in Surfactant Micelles Provides Platform for Membrane Structure StudiesSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
12/22/2016Neutrons Identify Oxygen Activation in LPMO enzymeNeutron Macromolecular Crystallography, X-ray Macromolecular CrystallographyAdvanced Photon Source, Spallation Neutron Source/High Flux Isotope ReactorMolecular Structure
05/23/2017Using Neutrons to Resolve Plasma Membrane Organization in Live BacteriaSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
08/03/2017Chemical Strategy for Conversion of CO2 to BiomassX-ray Macromolecular CrystallographyAdvanced Photon Source, Stanford Synchrotron Radiation LightsourceMolecular Structure
11/19/2020Enhancing Enzyme Catalysis Through Directed EvolutionX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
08/14/2020Phenoxy Radical-Radical Coupling in PlantsX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
06/22/2017Archaeal Abundance on Human Skin Changes with AgeSynchrotron Infrared Hyperspectral ImagingAdvanced Light SourceChemical and Elemental Information
08/11/2017Hacking the Bacterial Social NetworkX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
06/16/2017Engineering Better Plant Cultivars for Iron Uptake by Modifying the Iron Deficiency Response in Arabidopsis thalianaX-ray Fluorescence ImagingAdvanced Photon Source, National Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceChemical and Elemental Information
05/09/2017Probing S-layer Protein Structural Dynamics with SAXSSolution X-ray ScatteringStanford Synchrotron Radiation LightsourceMolecular Structure
09/08/2017The Origins of Photosynthesis in a Sun-Loving BacteriaX-ray Macromolecular CrystallographyAdvanced Light Source, Advanced Photon SourceMolecular Structure
08/14/2017X-ray Footprinting Solves Mystery of Metal-Breathing ProteinAdvanced Light SourceMolecular Structure
12/05/2016Designing Cyclic Oligomers: Greater than the Sum of Their PartsSolution X-ray Scattering, X-ray Macromolecular CrystallographyAdvanced Light Source, Advanced Photon SourceMolecular Structure
09/09/2017Measuring and Modeling Poplar Root Water Extraction After Drought Using Neutron ImagingNeutron ImagingSpallation Neutron Source/High Flux Isotope ReactorCell and Tissue Structure
06/30/2017Finding New Clues to a Common Respiratory VirusX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
11/02/2017Arsenic Accumulation in Benthic Periphytic BiofilmsX-ray Fluorescence ImagingStanford Synchrotron Radiation LightsourceChemical and Elemental Information
10/01/2016Crystal Clear Imaging Infrared Brings to Light Nanoscale Molecular ArrangementSynchrotron Infrared Hyperspectral ImagingAdvanced Light SourceChemical and Elemental Information
07/10/2017Novel Orange Carotenoid Proteins Shed Light on Evolution of Cyanobacteria PhotoprotectionX-ray Macromolecular CrystallographyAdvanced Light SourceMolecular Structure
01/11/2017Study Pinpoints Most Reactive Areas of Nanoscale ParticlesSynchrotron Infrared Hyperspectral ImagingAdvanced Light SourceChemical and Elemental Information
01/19/2017A Regional Model for Uranium Redox and MobilityX-ray Absorption and Emission SpectroscopyStanford Synchrotron Radiation LightsourceChemical and Elemental Information
04/07/2017Direct Measurement of Protein Dynamics In VivoSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
04/18/2017Could This Enzyme Help Turn Biofuel Waste into Something Useful?X-ray Macromolecular CrystallographyAdvanced Light SourceMolecular Structure
07/11/2017Brown Rot Fungi Reveal a New Approach for Biomass Conversion to Fuels and ChemicalsSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
09/19/2019Dynamics on Cellulose Show Two Important Populations from Neutron Scattering and SimulationsSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
11/15/2017Cyanobacterial Studies Examine Cellular Structure During Nitrogen StarvationSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
12/13/2017Binding of Alternative Substrates to NitrogenaseX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
01/17/2018"Zip Code" Mechanism for Hormone Signaling RevealedX-ray Macromolecular CrystallographyNational Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceMolecular Structure
01/18/2018Structure of a Flavoenzyme Assembly IntermediateX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
12/14/2017Using Plants to Immobilize and Stabilize Arsenic in the SoilX-ray Absorption and Emission Spectroscopy, X-ray Fluorescence ImagingNational Synchrotron Light Source II, Stanford Synchrotron Radiation LightsourceChemical and Elemental Information
08/30/2018Directed Evolution Mimics Allosteric Activation of EnzymesX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
08/21/2018Potent BacteriaX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
11/15/2018Controlled Self-Assembly of a Designed NucleoproteinSolution X-ray ScatteringStanford Synchrotron Radiation LightsourceMolecular Structure
02/05/2019Peptide Induced Lateral Segregation of Lipids in a BiomembraneSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
02/25/2019Breakthrough Shines Light on Disease-Fighting ProteinCryo-Electron Microscopy, X-ray Macromolecular CrystallographyAdvanced Photon Source, Stanford Synchrotron Radiation LightsourceMolecular Structure
02/18/2019An Efficient and Economical Strategy for the Design of Functional MetalloproteinsX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
02/22/2019DNA Gets a Bigger AlphabetX-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
03/05/2019Understanding Cadmium Distribution in Hyperaccumulating Plants for Efficient PhytoremediationX-ray Fluorescence ImagingStanford Synchrotron Radiation LightsourceChemical and Elemental Information
04/05/2019Influence of Chemically Disrupted Photosynthesis on Cyanobacterial Thylakoid DynamicsSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
04/12/2019What Enables the Fastest Carbon Dioxide Fixation by Reductive Carboxylase?X-ray Macromolecular CrystallographyStanford Synchrotron Radiation LightsourceMolecular Structure
05/03/2019New Approach for Solving Protein Structures from Tiny CrystalsX-ray Macromolecular CrystallographyNational Synchrotron Light Source IIMolecular Structure
07/10/2019Structuring Sweetness: What Makes Stevia 200 Times Sweeter than Sugar?X-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
07/10/2019Structural Characterization of Chitinases from Agave tequilanaSolution X-ray ScatteringStanford Synchrotron Radiation LightsourceMolecular Structure
11/19/2019How Essential Membrane Lipids Interact to Regulate Cellular ProcessesX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
11/29/2019Ammonia-Salt Solvent Promotes Cellulosic Biomass Deconstruction Under Ambient Processing ConditionsSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
12/09/2019Research at the Advanced Photon Source Leads to New Ebola DrugX-ray Macromolecular CrystallographyAdvanced Photon SourceMolecular Structure
08/29/2020How Proteins Remodel DNA in Bacteria Under StressSolution X-ray Scattering, X-ray Macromolecular Crystallography, Soft X-ray TomographyAdvanced Light SourceMolecular Structure, Cell and Tissue Structure
08/13/2020Cell Membrane Proteins Imaged in 3DX-ray Fluorescence ImagingAdvanced Photon Source, National Synchrotron Light Source IIChemical and Elemental Information
07/21/2020Role of Solvents in Efficient Biomass DeconstructionSmall-Angle Neutron ScatteringSpallation Neutron Source/High Flux Isotope ReactorMolecular Structure
02/14/2020How Dinosaur Blood Vessels Preserve Through the AgesSynchrotron Infrared Hyperspectral ImagingAdvanced Light SourceChemical and Elemental Information
06/23/2020Novel Cell Membrane Model Could Be Key to Uncovering New Protein PropertiesCryo-Electron Microscopy, Small-Angle Neutron Scattering, Solution X-ray ScatteringNational Synchrotron Light Source II, Spallation Neutron Source/High Flux Isotope ReactorMolecular Structure, Cell and Tissue Structure
10/07/2020"Missing Link" in Evolutionary History of the Carbon-Fixing Protein RubiscoSolution X-ray ScatteringAdvanced Light SourceMolecular Structure