Synchrotron Infrared Hyperspectral Imaging

Images of chemical events and metabolic function within a sample

Symbiotic associations in the rhizosphere influence the distribution of nutrients that promote the growth and development of both plants and microbes. (A) Fourier-transform infrared imaging (FTIR), a common infrared hyperspectral imaging technique, shows evidence of symbiotic exchange of nitrate and sucrose between a poplar root (gray) and fungi in the poplar rhizosphere (green). (B) Nitrate concentration is elevated in regions of high fungal colonization close to the root (faint black line at top). As distance from the root increases (white arrow), the fungal population and nitrate levels decrease. (C) Sucrose concentration, on the other hand, increases with distance from the root (faint black line). These trends suggest symbiotic sharing of nutrients between plant and fungi, where the fungi consume sucrose and sequester nitrate for the plant’s use. [Reprinted with permission from Victor, T., et al. 2017. “Imaging Nutrient Distribution in the Rhizosphere Using FTIR Imaging,” Analytical Chemistry 89(9) 4831–37. DOI:10.1021/acs.analchem.6b04376. Copyright 2017 American Chemical Society.]

A widely used method of infrared hyperspectral imaging is Fourier-transform infrared imaging (FTIR), or FTIR spectromicroscopy. This technique correlates infrared maps with visible microscopy images to map the distributions of molecular compositions of interest within biological samples and to reveal sample morphology and structure. Compared to desktop FTIR chemical imaging techniques, synchrotron-based FTIR (sFTIR) can provide detailed images of the location and concentration of molecules with a resolution of ~2.0 to ~10 µm per pixel and a sensitivity 100-X to 1,000-X.

The BER-supported sFTIR resource at Lawrence Berkeley National Laboratory’s Advanced Light Source also has multiple time-resolved microfluidic sFTIR imaging capabilities for tracking the location and dynamics of biochemical processes in live cells or biofilms. This can be applied to study chemical events in real time on a timescale of seconds and longer. Sample requirements and preparation are generally minimal, enabling diverse user experiments and the study of wide-ranging science questions.

Key Features of sFTIR Imaging

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  Bacterial components in an algal system might play relevant roles in algal plasticity and adaptive responses to a changing environment. Synchrotron-based Fourier-transform infrared spectromicroscopy (sFTIR) reveals the distributions of dissolution-resistant, high-magnesium carbonate minerals and other compounds involved in biomineralization inside a single cell of a marine coralline algae. This suggests diverse metabolic function by symbiotic microbes which may help confer resilience to ocean acidification. [Reprinted under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International. https://creativecommons.org/licenses/by-nc-nd/4.0/ from Valdespino-Castillo, P. M., et al. 2021. “Interplay of Microbial Communities with Mineral Environments in Coralline Algae,” Science of The Total Environment 757, 143877. DOI: 10.1016/j.scitotenv.2020.143877.]

  Enables chemical mapping of functional groups of molecules.
• Chemical information is restricted to “infrared active” molecules.
• Spatial resolution ranges from 2.0 to 10 µm to match research interests.
• Nano-sFTIR provides chemical information in the 4,000 to 650 cm^-1 spectral range with a spatial resolution of ~20 nm.
• The low energy range (<500 meV) of the infrared beam enables non-invasive tracking of chemical events over time.
• Accommodates diverse sample types including cells, organisms, soil, rock, and experimental devices.
• Enables simultaneous data collection about the functional groups of multiple molecules (e.g., polysaccharides, lipids, minerals)

BER Researchers Use sFTIR Imaging to Study:

• Microbes that promote plant growth (Pantoea sp. YR343)
• Terrestrial soil systems (soil organic matter stability and persistence) in different climate zones
• Biogeochemical cycles in permafrost ecosystems
• Nanoscale characterization of released vesicles
• Biogenesis of minerals
• Bioenergy (e.g., consolidated bioprocessing)

See more examples in the Research Highlights table below.

Sample Considerations

• Sample environments range from ambient air for aerobic microbes to oxygen-free for anaerobic microorganisms.
• Sample topography is preferably no greater than 10 μm for sFTIR spectromicroscopy, but less than 0.1 μm for Synchrotron Infrared Nano-spectroscopy (SINS) imaging.
• Samples can be any thickness, but infrared ray penetration of a biological or biogeochemical sample is generally less than 20 μm.
• Samples must stay flat when mounted under the beam. During analysis, the sample will move, not the beam.
• Samples the size of a standard microscope slide are generally suitable for infrared spectromicroscopy. Discuss unique sample dimensions and properties with beamline experts.

Infrared 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 links below.

• Berkeley Synchrotron Infrared Structural Biology Imaging Program (BSISB) — Advanced Light Source

Research Highlights