Label-Free Multiphoton Imaging of Microbes in Root, Mineral, and Soil Matrices with Time-Gated Coherent Raman and Fluorescence Lifetime Imaging.

Imaging biogeochemical interactions in complex microbial systems─such as those at the soil-root interface─is crucial to studies of climate, agriculture, and environmental health but complicated by the three-dimensional (3D) juxtaposition of materials with a wide range of optical properties. We developed a label-free multiphoton nonlinear imaging approach to provide contrast and chemical information for soil microorganisms in roots and minerals with epi-illumination by simultaneously imaging two-photon excitation fluorescence (TPEF), coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and sum-frequency mixing (SFM). We used fluorescence lifetime imaging (FLIM) and time gating to correct CARS for the autofluorescence background native to soil particles and fungal hyphae (TG-CARS) using time-correlated single-photon counting (TCSPC). We combined TPEF, TG-CARS, and FLIM to maximize image contrast for live fungi and bacteria in roots and soil matrices without fluorescence labeling. Using this instrument, we imaged symbiotic arbuscular mycorrhizal fungi (AMF) structures within unstained plant roots in 3D to 60 µm depth. High-quality imaging was possible at up to 30 µm depth in a clay particle matrix and at 15 µm in complex soil preparation. TG-CARS allowed us to identify previously unknown lipid droplets in the symbiotic fungus, Serendipita bescii. We also visualized unstained putative bacteria associated with the roots of Brachypodium distachyon in a soil microcosm. Our results show that this multimodal approach holds significant promise for rhizosphere and soil science research.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Increasing aggregate size reduces single-cell organic carbon incorporation by hydrogel-embedded wetland microbes.

Microbial degradation of organic carbon in sediments is impacted by the availability of oxygen and substrates for growth. To better understand how particle size and redox zonation impact microbial organic carbon incorporation, techniques that maintain spatial information are necessary to quantify elemental cycling at the microscale. In this study, we produced hydrogel microspheres of various diameters (100, 250, and 500 µm) and inoculated them with an aerobic heterotrophic bacterium isolated from a freshwater wetland (Flavobacterium sp.), and in a second experiment with a microbial community from an urban lacustrine wetland. The hydrogel-embedded microbial populations were incubated with 13C-labeled substrates to quantify organic carbon incorporation into biomass via nanoSIMS. Additionally, luminescent nanosensors enabled spatially explicit measurements of oxygen concentrations inside the microspheres. The experimental data were then incorporated into a reactive-transport model to project long-term steady-state conditions. Smaller (100 µm) particles exhibited the highest microbial cell-specific growth per volume, but also showed higher absolute activity near the surface compared to the larger particles (250 and 500 µm). The experimental results and computational models demonstrate that organic carbon availability was not high enough to allow steep oxygen gradients and as a result, all particle sizes remained well-oxygenated. Our study provides a foundational framework for future studies investigating spatially dependent microbial activity in aggregates using isotopically labeled substrates to quantify growth.

Microbiome processing of organic nitrogen input supports growth and cyanotoxin production of Microcystis aeruginosa cultures.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

Single-cell isotope tracing reveals functional guilds of bacteria associated with the diatom Phaeodactylum tricornutum.

Bacterial remineralization of algal organic matter fuels algal growth but is rarely quantified. Consequently, we cannot currently predict whether some bacterial taxa may provide more remineralized nutrients to algae than others. Here, we quantified bacterial incorporation of algal-derived complex dissolved organic carbon and nitrogen and algal incorporation of remineralized carbon and nitrogen in fifteen bacterial co-cultures growing with the diatom Phaeodactylum tricornutum at the single-cell level using isotope tracing and nanoSIMS. We found unexpected strain-to-strain and cell-to-cell variability in net carbon and nitrogen incorporation, including non-ubiquitous complex organic nitrogen utilization and remineralization. We used these data to identify three distinct functional guilds of metabolic interactions, which we termed macromolecule remineralizers, macromolecule users, and small-molecule users, the latter exhibiting efficient growth under low carbon availability. The functional guilds were not linked to phylogeny and could not be elucidated strictly from metabolic capacity as predicted by comparative genomics, highlighting the need for direct activity-based measurements in ecological studies of microbial metabolic interactions.

Application of modern autoradiography to nuclear forensic analysis.

Modern autoradiography techniques based on phosphorimaging technology using image plates (IPs) and digital scanning can identify heterogeneities in activity distributions and reveal material properties, serving to inform subsequent analyses. Here, we have adopted these advantages for applications in nuclear forensics, the technical analysis of radioactive or nuclear materials found outside of legal control to provide data related to provenance, production history, and trafficking route for the materials. IP autoradiography is a relatively simple, non-destructive method for sample characterization that records an image reflecting the relative intensity of alpha and beta emissions from a two-dimensional surface. Such data are complementary to information gathered from radiochemical characterization via bulk counting techniques, and can guide the application of other spatially resolved techniques such as scanning electron microscopy (SEM) and secondary ion mass spectrometry (SIMS). IP autoradiography can image large 2-dimenstional areas (up to 20×40cm), with relatively low detection limits for actinides and other radioactive nuclides, and sensitivity to a wide dynamic range (105) of activity density in a single image. Distributions of radioactivity in nuclear materials can be generated with a spatial resolution of approximately 50µm using IP autoradiography and digital scanning. While the finest grain silver halide films still provide the best possible resolution (down to ∼10µm), IP autoradiography has distinct practical advantages such as shorter exposure times, no chemical post-processing, reusability, rapid plate scanning, and automated image digitization. Sample preparation requirements are minimal, and the analytical method does not consume or alter the sample. These advantages make IP autoradiography ideal for routine screening of nuclear materials, and for the identification of areas of interest for subsequent micro-characterization methods. In this paper we present a summary of our setup, as modified for nuclear forensic sample analysis and related research, and provide examples of data from select samples from the nuclear fuel cycle and historical nuclear test debris.