Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Abstract

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of classes of biomolecules (26, 43; X. Zhang, T. Araki, A. P. Hitchcock, J. R. Lawrence, and G. G. Leppard, unpublished data).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...

Speciation and Quantitative Mapping of Metal Species in Microbial Biofilms Using Scanning Transmission X-ray Microscopy

“…These observations are in keeping with those obtained by confocal microscopies of similarly grown river biofilms (17). The area selected for detailed examination was chosen on the basis of large area survey difference maps (I709 eV-I702 eV) which quickly identify areas of high metal content.…”

“…STXM can provide quantitative maps of chemical species at environmentally relevant concentrations (i.e., mg/kg), with a spatial resolution of better than 50 nm (15)(16)(17). Full field soft X-ray transmission microscopy has been used to examine Pseudomonas putida biofilms (18).…”

Correlative electron and X-ray microscopy: probing chemistry and bonding with high spatial resolution

“…Detailed studies of live cells using soft X-ray or electron microscopy have not been carried out to date and preliminary investigations indicate that radiation damage is a critical issue in unfixed biological samples, leading to state, and show how STXM-XAS may be used to gain more detailed spectroscopic information from hydrated biological samples than the water-window imaging technique. 66,72 Dynes et al 73 demonstrated that it is possible to perform correlative optical microscopy, TEM and STXM on a single 25 sample by using an elegant labelling technique. In this work, biofilm sections were labelled with CdSe/ZnS quantum dots designed to bind to polysaccharides.…”

“…Lawrence et al, 66 used a range of fluorescent markers to stain nucleic acids, proteins, lectins and lipids within a river biofilm sample (figure 7). Confocal laser scanning microscopy (CLSM) 35 was then used to map these components across hydrated samples contained in silicon nitride 'wet cells' 74 with spatial resolution of < 1 μm.…”

Synchrotron‐Based Near‐Edge X‐Ray Spectroscopy of Natural Organic Matter in Soils and Sediments

“…The other peak assignments primarily come from data presented in Myneni (2002), and they remain unconfirmed and are contradicted in some cases. For example, Lawrence et al (2003) list a lipid O-NEXAFS peak at 534 eV, which should according to Table 17.6 be due to alcohol groups. The presence of large water peaks on NOM can obscure the presence of ether moieties and can also preclude the use of combined carbon, nitrogen, and oxygen NEXAFS to measure NOM elemental ratios.…”