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.

• National Center for X-Ray Tomography (Beamline 2.1) — Advanced Light Source

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