Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highestresolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.Scanning transmission X-ray microscopy (STXM) (1, 2), which uses near-edge X-ray absorption spectroscopy (NEXAFS) as its contrast mechanism, is a powerful new tool that can be applied to fully hydrated biological materials. This is possible due to the ability of soft X rays to penetrate water, the presence of suitable analytical core edges in the soft X-ray region, and reduced radiation damage (compared to that caused by electron beam techniques). Soft X rays also provide spatial resolution of better than 50 nm, which is suitable for imaging bacteria and bacterial biofilms. The spectral resolution is on the order of 100 meV, which in combination with their intrinsic spectral properties is sufficient to provide good differentiation of classes of biomolecules (26, 43; X. Zhang, T. Araki, A. P. Hitchcock, J. R. Lawrence, and G. G. Leppard, unpublished data). Through the application of tunable soft X rays and appropriate analysis of X-ray absorption spectra in the form of NEXAFS image sequences (15), quantitative chemical mapping at a spatial scale below 50 nm may be achieved. With use of the appropriate spectral range, NEXAFS microscopy provides detailed, quantitative speciation and elemental analysis with parts-per-thousand local and parts-per-million global sensitivities with transmission detection. Soft X-ray microscopy provides a combination of suitable spatial resolution and chemical information at a microscale. In addition, soft X rays interact with nearly all elements and also allow mapping of chemical species based on bonding structure (2). Further, the method uses the intrinsic X-ray absorption properties of the sample, thus eliminating the need for addition of reflective, absorptive, or fluorescent probes and markers that may introduce artifacts or complicate interpretation. It is...
Nephila clavipes dragline silk microstructure has been investigated by scanning transmission X-ray microscopy (STXM), a technique that allows quantitative mapping of the level of orientation of the peptide groups at high spatial resolution (<50 nm). Maps of the orientation parameter P2 have been derived for spider silk for the first time. Dragline silk presents a very fine microstructure in which small, highly oriented domains (average area of 1800 nm2, thus clearly bigger than individual beta-sheet crystallites) are dispersed in a dominant, moderately oriented matrix with several small unoriented domains. Our results also highlight the orientation of the noncrystalline fraction in silk, which has been underestimated in numerous structural models. No evidence of either a regular lamellar structure or any periodicity along the fiber was observed at this spatial resolution. The surface of fresh spider silk sections consists of a approximately 30-120 nm thick layer of highly oriented protein chains, which was found to vary with the reeling speed, where web building (0.5 cm/s) and lifeline (10 cm/s) spinning speeds were investigated. While the average level of orientation of the protein chains is unaffected by the spinning speed, STXM measurements clearly highlight microstructure differences. The slowpull fiber contains a larger fraction of highly oriented domains, while the protein chains are more homogeneously oriented in the fastpull fiber. In comparison, cocoon silk from the silkworm Bombyx mori presents a narrower orientation distribution. The strength-extensibility combination found in spider dragline silk is associated with its broad orientation distribution of highly interdigitated and unoriented domains.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.