Heavy water and 15N labelling with NanoSIMS analysis reveals growth rate‐dependent metabolic heterogeneity in chemostats

To measure single‐cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and ¹⁵N ammonium in combination with multi‐isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single‐cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single‐cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (～0.1 atom % ²H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (～6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.     

3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.     

Unraveling uncultivable pesticide degraders via stable isotope probing (SIP)

Uncultivable microorganisms account for over 99% of all species on earth, playing essential roles in ecological processes such as carbon/nitrogen cycle and chemical mineralization. Their functions remain unclear in ecosystems and natural habitats, requiring cutting-edge biotechnologies for a deeper understanding. Stable isotope probing (SIP) incorporates isotope-labeled elements, e.g. ^13?C, ^18?O or ^15?N, into the cellular components of active microorganisms, serving as a powerful tool to link phylogenetic identities to their ecological functions in situ. Pesticides raise increasing attention for their persistence in the environment, leading to severe damage and risks to the ecosystem and human health. Cultivation and metagenomics help to identify either cultivable pesticide degraders or potential pesticide metabolisms within microbial communities, from various environmental media including the soil, groundwater, activated sludge, plant rhizosphere, etc. However, the application of SIP in characterizing pesticide degraders is limited, leaving considerable space in understanding the natural pesticide mineralization process. In this review, we try to comprehensively summarize the fundamental principles, successful cases and technical protocols of SIP in unraveling functional-yet-uncultivable pesticide degraders, by raising its shining lights and shadows. Particularly, this study provides deeper insights into various feasible isotope-labeled substrates in SIP studies, including pesticides, pesticide metabolites, and similar compounds. Coupled with other techniques, such as next-generation sequencing, nanoscale secondary ion mass spectrometry (NanoSIMS), single cell genomics, magnetic-nanoparticle-mediated isolation (MMI) and compound-specific isotope analysis (CSIA), SIP will significantly broaden our understanding of pesticide biodegradation process in situ.     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

Quantification of Tetradesmus obliquus (Chlorophyceae) cell size and lipid content heterogeneity at single‐cell level

Much of our current knowledge of microbial growth is obtained from studies at a population level. Driven by the realization that processes that operate within a population might influence a population's behavior, we sought to better understand Tetradesmus obliquus (formerly Scenedesmus obliquus ) physiology at the cellular level. In this work, an accurate pretreatment method to quantitatively obtain single cells of T. obliquus , a coenobia‐forming alga, is described. These single cells were examined by flow cytometry for triacylglycerol (TAG ), chlorophyll, and protein content, and their cell sizes were recorded by coulter counter. We quantified heterogeneity of size and TAG content at single‐cell level for a population of T. obliquus during a controlled standard batch cultivation. Unexpectedly, variability of TAG content per cell within the population increased throughout the batch run, up to 400 times in the final stage of the batch run, with values ranging from 0.25 to 99 pg · cell^?1. Two subpopulations, classified as having low or high TAG content per cell, were identified. Cell size also increased during batch growth with average values from 36 to 70 μm³ · cell^?1; yet cell size variability increased only up to 16 times. Cell size and cellular TAG content were not correlated at the single‐cell level. Our data show clearly that TAG production is affected by cell‐to‐cell variation, which suggests that its control and better understanding of the underlying processes may improve the productivity of T. obliquus for industrial processes such as biodiesel production.     

Scanning transmission X-ray microscopy study of microbial calcification

Calcium phosphates and calcium carbonates are among the most prevalent minerals involved in microbial fossilization. Characterization of both the organic and mineral components in biomineralized samples is, however, usually difficult at the appropriate spatial resolution (i.e. at the submicrometer scale). Scanning transmission X‐ray microscopy (STXM) was used to measure C K‐edge, P L‐edge, and Ca L‐edge near‐edge X‐ray absorption fine structure (NEXAFS) spectra of some calcium‐containing minerals common in biomineralization processes and to study the experimental biomineralization by the model microorganism, Caulobacter crescentus. We show that the Ca L_2,3‐edges for hydroxyapatite, calcite, vaterite, and aragonite are unique and can be used as probes to detect these different mineral phases. Using these results, we showed that C. crescentus cells, when cultured in the presence of high calcium concentration, precipitated carbonate hydroxyapatite. In parallel, we detected proteins, polysaccharides, and nucleic acids in the mineralizing bacteria at the single‐cell scale. Finally, we discussed the utility of STXM for the study of natural fossilized microbial systems.     

Micro‐ to nanostructure and geochemistry of extant crinoidal echinoderm skeletons

This paper reports the results of micro‐ to nanostructural and geochemical analyses of calcitic skeletons from extant deep‐sea stalked crinoids. Fine‐scale (SEM, FESEM, AFM) observations show that the crinoid skeleton is composed of carbonate nanograins, about 20–100 nm in diameter, which are partly separated by what appears to be a few nm thick organic layers. Sub‐micrometre‐scale geochemical mapping of crinoid ossicles using a NanoSIMS ion microprobe, combined with synchrotron high‐spatial‐resolution X‐ray micro‐fluorescence (μ‐XRF) maps and X‐ray absorption near‐edge structure spectroscopy (XANES) show that high Mg concentration in the central region of the stereom bars correlates with the distribution of S‐sulphate, which is often associated with sulphated polysaccharides in biocarbonates. These data are consistent with biomineralization models suggesting a close association between organic components (including sulphated polysaccharides) and Mg ions. Additionally, geochemical analyses (NanoSIMS, energy dispersive spectroscopy) reveal that significant variations in Mg occur at many levels: within a single stereom trabecula, within a single ossicle and within a skeleton of a single animal. Together, these data suggest that physiological factors play an important role in controlling Mg content in crinoid skeletons and that great care should be taken when using their skeletons to reconstruct, for example, palaeotemperatures and Mg/Ca palaeo‐variations of the ocean.     

Integrated co‐regulation of bacterial arsenic and phosphorus metabolisms

Arsenic ranks first on the US Environmental Protection Agency Superfund List of Hazardous Substances. Its mobility and toxicity depend upon chemical speciation, which is significantly driven by microbial redox transformations. Genome sequence‐enabled surveys reveal that in many microorganisms genes essential to arsenite (AsIII) oxidation are located immediately adjacent to genes coding for functions associated with phosphorus (Pi) acquisition, implying some type of functional importance to the metabolism of As, Pi or both. We extensively document how expression of genes key to AsIII oxidation and the Pi stress response are intricately co‐regulated in the soil bacterium Agrobacterium tumefaciens. These observations significantly expand our understanding of how environmental factors influence microbial AsIII metabolism and contribute to the current discussion of As and P metabolism in the microbial cell.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM,TEM/X-EDS,and NanoSIMS

Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1 nm was used for imaging the ultrastructure of algae cells which have diameters of 5–10 μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1 μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200 nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50 nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast.     

New approaches in bioprocess‐control: Consortium guidance by synthetic cell‐cell communication based on fungal pheromones

Bioconversions in industrial processes are currently dominated by single‐strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single‐strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium‐based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell–cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone‐responsive gene expression, thereby creating pheromone‐dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone‐controlled sensor‐actor systems, exploiting their unique metabolic properties for microbial consortia approaches.     

Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single‐cell precision to define cell identities in a large and heterogeneous population of cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi‐ATOM, based upon ultrafast phase‐gradient encoding, outperforms state‐of‐the‐art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi‐ATOM for large‐scale, label‐free, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi‐ATOM could empower new strategies in large‐scale biophysical single‐cell analysis with applications in biology and enriching disease diagnostics.      

A critical review of NanoSIMS in analysis of microbial metabolic activities at single-cell level

Over 3.8 billion years of evolution has enabled many microbial species a versatile metabolism. However, limited by experimental methods, some unique metabolism remains unknown or unclear. A major obstacle is to attribute the incorporation of certain nutrients into a noncultivable species out of a complex microbial community. Such difficulty could be solved if we are able to directly observe substrate uptake at the single-cell level. Nanoscale secondary ion mass spectrometry (NanoSIMS) is a powerful tool for revealing element distribution in nanometer-scale resolution in the fields such as material sciences, geosciences and astronomy. In this review, we focus on another applicability of NanoSIMS in microbiology. In such fields, physiological properties and metabolic activities of microorganisms can be revealed with a single-cell scale resolution by NanoSIMS solely or in combination with other techniques. This review will highlight the features of NanoSIMS in analyzing the metabolic activities of carbon, nitrogen, metal irons by mixed-culture assemblies. Some values of NanoSIMS in environmental microbiology are expected to be discussed via this review.     

Environmentally controlled bacterial vesicle‐mediated export

Over the past two decades, researchers studying both microbial and host cell communities have gained an appreciation for the ability of bacteria to produce, regulate, and functionally utilize outer membrane vesicles (OMVs) as a means to survive and interact with their cellular and acellular environments. Common ground has emerged, as it appears that vesicle production is an environmentally controlled and specific secretion process; however, it has been challenging to discover the principles that govern fundamentals of vesicle‐mediated transport. Namely, there does not appear to be a single mechanism modulating OMV export, nor universal “markers” for OMV cargo incorporation, nor particular host cell responses common to treatment with all OMVs. Given the diversity of species studied, their differences in envelope architecture and composition, the diversity of environmentally regulated bacterial processes, and the variety of interactions between bacteria and their abiotic and biotic environments, this is hardly surprising. Nevertheless, the ability of bacteria to control exported material in the context of a packaged insoluble particle, a vesicle, is emerging as a significant contribution to bacterial viability, biofilm communities, and bacterial‐host interactions. In this review, we focus on detailing important, recent findings regarding the content and functional differences in bacterially secreted vesicles that are influenced by growth conditions.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

Spatial cycles in G‐protein crowd control

The nature of living systems and their apparent resilience to the second law of thermodynamics has been the subject of extensive investigation and imaginative speculation. The segregation and compartmentalization of proteins is one manifestation of this departure from equilibrium conditions; the effect of which is now beginning to be elucidated. This should not come as a surprise, as even a cursory inspection of cellular processes reveals the large amount of energetic cost borne to maintain cell‐scale patterns, separations and gradients of molecules. The G‐proteins, kinases, calcium‐responsive proteins have all been shown to contain reaction cycles that are inherently coupled to their signalling activities. G‐proteins represent an important and diverse toolset used by cells to generate cellular asymmetries. Many small G‐proteins in particular, are dynamically acylated to modify their membrane affinities, or localized in an activity‐dependent manner, thus manipulating the mobility modes of these proteins beyond pure diffusion and leading to finely tuned steady state partitioning into cellular membranes. The rates of exchange of small G‐proteins over various compartments, as well as their steady state distributions enrich and diversify the landscape of possibilities that GTPase‐dependent signalling networks can display over cellular dimensions. The chemical manipulation of spatial cycles represents a new approach for the modulation of cellular signalling with potential therapeutic benefits.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Oufti: an integrated software package for high‐accuracy,high‐throughput quantitative microscopy analysis

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.