Molecular imaging of plant-microbe interactions on the Brachypodium seed surface.

Plant growth-promoting rhizobacteria (PGPR) play a crucial role in biological control and pathogenic defense on and within plant tissues, however the mechanisms by which plants associate with PGPR to elicit such beneficial effects need further study. Here, we present time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of Brachypodium distachyon (Brachypodium) seeds with and without exposure to two model PGPR, i.e., Gram-negative Pseudomonas fluorescens SBW25 (P.) and Gram-positive Arthrobacter chlorophenolicus A6 (A.). Delayed image extraction was used to image PGPR-treated seed sections to reveal morphological changes. ToF-SIMS spectral comparison, principal component analysis (PCA), and two-dimensional (2D) imaging show that the selected PGPR have different effects on the host seed surface, resulting in changes in chemical composition and morphology. Metabolite products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and indole-3-acetic acid (IAA), were identified on the PGPR-treated seed surfaces. These compounds have different distributions on the Brachypodium seed surface for the two PGPR, indicating that the different bacteria elicit distinct responses from the host. Our results illustrate that ToF-SIMS is an effective tool to study plant-microbe interactions and to provide insightful information with submicrometer lateral resolution of the chemical distributions associated with morphological features, potentially offering a new way to study the mechanisms underlying beneficial roles of PGPR.

Correlative surface imaging reveals chemical signatures for bacterial hotspots on plant roots.

The rhizosphere is arguably the most complex microbial habitat on Earth, comprising an integrated network of plant roots, soil and a highly diverse microbial community (the rhizosphere microbiome). Understanding, predicting and controlling plant-microbe interactions in the rhizosphere will allow us to harness the plant microbiome as a means to increase or restore plant ecosystem productivity, improve plant responses to a wide range of environmental perturbations, and mitigate the effects of climate change by designing ecosystems for long-term soil carbon storage. To this end, it is imperative to develop new molecular approaches with high spatial resolution to capture interactions at the plant-microbe, microbe-microbe, and plant-plant interfaces. In this work, we designed an imaging sample holder that allows integrated surface imaging tools to map the same locations of a plant root-microbe interface with submicron lateral resolutions, providing novel in vivo analysis of root-microbe interactions. Specifically, confocal fluorescence microscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used for the first time for the correlative imaging of the Brachypodium distachyon root and its interaction with Pseudomonas SW25, a typical plant growth-promoting soil bacterium. Imaging data suggest that the root surface is inhomogeneous and that the interaction between Pseudomonas and Brachypodium roots was confined to only a few spots along the sampled root segments and that the bacterial attachment spots were enriched in Na- and S-related and high-mass organic species. We conclude that the attachment of the Pseudomonas cells to the root surface is outcompeted by strong root-soil mineral interactions but facilitated by the formation of extracellular polymeric substances (EPS).

Probing sulphur clusters in a microfluidic electrochemical cell with synchrotron-based photoionization mass spectrometry.

We present synchrotron-based mass spectrometry to probe products formed in a lithium sulphide electrolyte. In operando analysis was carried out at two different potentials in a vacuum compatible microfluidic electrochemical cell. Mass spectral observations show that the charged electrolyte formed sulphur clusters under dynamic conditions, demonstrating electrolyte electron shuttling.

Mass spectral imaging showing the plant growth-promoting rhizobacteria's effect on the Brachypodium awn.

The plant growth-promoting rhizobacteria (PGPR) on the host plant surface play a key role in biological control and pathogenic response in plant functions and growth. However, it is difficult to elucidate the PGPR effect on plants. Such information is important in biomass production and conversion. Brachypodium distachyon (Brachypodium), a genomics model for bioenergy and native grasses, was selected as a C3 plant model; and the Gram-negative Pseudomonas fluorescens SBW25 (P.) and Gram-positive Arthrobacter chlorophenolicus A6 (A.) were chosen as representative PGPR strains. The PGPRs were introduced to the Brachypodium seed's awn prior to germination, and their possible effects on the seeding and growth were studied using different modes of time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements, including a high mass-resolution spectral collection and delayed image extraction. We observed key plant metabolic products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and auxin indole-3-acetic acid in the Brachypodium awns. Furthermore, principal component analysis and two-dimensional imaging analysis reveal that the Brachypodium awns are sensitive to the PGPR, leading to chemical composition and morphology changes on the awn surface. Our results show that ToF-SIMS can be an effective tool to probe cell-to-cell interactions at the biointerface. This work provides a new approach to studying the PGPR effects on awn and shows its potential for the research of plant growth in the future.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

Recent developments in the synthesis, properties, and biomedical applications of core/shell superparamagnetic iron oxide nanoparticles with gold.

In the last decade, magnetic nanoparticles (MNPs), especially superparamagnetic iron oxide nanoparticles (SPIONs), have immensely promoted the advancement of diagnostics and theranostics in the biomedical field. The unique properties of the SPIONs-core and the functional gold (Au)-shell together (SPIONS/Au core/shell or CS) have a wide range of biomedical applications including, but not limited to, magnetic resonance imaging (MRI), dual modality MRI/computed tomography (CT), photo-induced and magnetic fluid hyperthermia (MFH), drug delivery, biosensors, and bio-separation. Researchers have made much effort to develop synthesis strategies for size control and surface modifications to achieve the desired properties of these CSs for applications in in vitro and in vivo studies. This review highlights recent developments in the synthesis and biomedical applications of SPIONs/Au CSs, including γ-Fe2O3/Au (maghemite), Fe3O4/Au (magnetite), and MFe2O4/Au (M = divalent metal ions) in the past seven years. More importantly, current trends of SPIONs/Au in relation to the biochemical industry are surveyed. Finally, we outline the developmental needs of SPIONs/Au from the perspective of material synthesis and their novel applications in disease diagnosis and treatment in the near future.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM.

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

Characterization of syntrophic Geobacter communities using ToF-SIMS.

The aggregation of syntrophic Geobacter metallireducens and Geobacter sulfurreducens is beneficial for enhancing direct interspecies electron transfer (DIET). Although DIET was suspected to occur on the microbial community surface, the surface chemical speciation of such cocultured communities remains unclear. In order to better understand surface interactions related to DIET, the authors characterized a series of samples associated with syntrophic G. metallireducens and G. sulfurreducens using surface sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis was used in spectral analysis. Our results show that the syntrophic Geobacter aggregates are significantly different from their planktonic cells, indicating a distinct chemical composition (i.e., amino acids, fatty acids, and lipids) and structure formed on their surface. Among these characteristic components, amino acid fragments dominated in the variance, suggesting the importance of proteins in the coculture. Additionally, the quorum sensing signal molecule N-butyryl-l-homoserine lactone was observed in cocultured Geobacter aggregates, implying its role in syntrophic growth and aggregate formation. Furthermore, the electron acceptor organism G. sulfurreducens was shown to be the dominant species in syntrophic communities that drove the syntrophic growth. These results demonstrate that unique chemical compositions distinguish syntrophic Geobacter aggregates from planktonic cells and suggest that ToF-SIMS may be a promising tool to understand the syntrophic mechanism and investigate interspecies electron transfer pathways in complex biofilms.