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A broad-spectrum fluorescent probe, which can be applied to monitoring H2S in various biological systems, has been rationally designed and synthesized. This specific probe was applied to localize the endogenous H2S in living Raw264.7 macrophage cells, HepG2 cells, and H9C2 cells. At the same time, the probe has successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS and CSE activity in H9C2 cells. This probe could serve as a powerful molecular imaging tool to further explore the physiological function and the molecular mechanisms of endogenous H2S in living animal systems.

We report the design and performance of a 3-electrode device for real time in situ scanning transmission X-ray microscopy studies of electrochemical processes under both static (sealed, non-flow) conditions and with a continuous flow of electrolytes. The device was made using a combination of silicon microfabrication and 3D printing technologies. The performance is illustrated by results of a study of copper deposition and stripping at a gold working electrode. X-ray absorption spectromicroscopy at the Cu 2p edge was used to follow the evolution as a function of potential and time of the spatial distributions of Cu(0) and Cu(i) species electro-deposited from an aqueous solution of copper sulphate. The results are interpreted in terms of competing mechanisms for the reduction of Cu(ii).

Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy.

Two novel fluorescein-based OCl − chemosensors were designed, synthesized and characterized. Both fluorescence probes utilize an irreversible OCl − -promoted oxidation reaction to trigger the activation of fluorescence. These probes were shown to be highly selective for OCl − , and showed real time responses as well as a positive linear relationship to OCl − concentrations. The probes were successfully applied for the detection of OCl − in tap water and for fluorescence imaging of OCl − in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell–EPS–mineral aggregates.

Speciation and quantitative mapping of elements, organic and inorganic compounds, and mineral phases in environmental samples at high spatial resolution is needed in many areas of geobiochemistry and environmental science. Scanning transmission X-ray microscopes (STXMs) provide a focused beam which can interrogate samples at a fine spatial scale. Quantitative chemical information can be extracted using the transmitted and energy-resolved X-ray fluorescence channels simultaneously. Here we compare the relative merits of transmission and low-energy X-ray fluorescence detection of X-ray absorption for speciation and quantitative analysis of the spatial distribution of arsenic(V) within cell-mineral aggregates formed by Acidovorax sp. strain BoFeN1, an anaerobic nitrate-reducing Fe(II)-oxidizing β-proteobacteria isolated from the sediments of Lake Constance. This species is noted to be highly tolerant to high levels of As(V). Related, As-tolerant Acidovorax-strains have been found in As-contaminated groundwater wells in Bangladesh and Cambodia wherein they might influence the mobility of As by providing sorption sites which might have different properties as compared to chemically formed Fe-minerals. In addition to demonstrating the lower detection limits that are achieved with X-ray fluorescence relative to transmission detection in STXM, this study helps to gain insights into the mechanisms of As immobilization by biogenic Fe-mineral formation and to further the understanding of As-resistance of anaerobic Fe(II)-oxidizing bacteria.

article i nfo Soft X-ray scanning transmission X-ray microscopy (STXM) was used to measure the Fe L2, 3 X-ray magnetic circular dichroism (XMCD) signal from individual, 30 nm diameter magnetosomes in a magnetotactic bacterium, specifically the marine vibrio strain MV-1. The Fe L2, 3 spectra recorded with circularly polarized X-rays from an elliptically polarizing undulator were very similar in shape and magnitude to those of fully saturated magnetite (Fe3O4). As previously determined by other techniques, our results show that the magnetic moments of individual magnetosomes arranged in linear chains in intact cells of strain MV-1 are all oriented in the same direction. The magnitude of the XMCD signal averaged over a chain of 9 magnetosomes in one cell is similar to that of single crystal magnetite. The spectral shape is slightly different, and indicates that magnetosomes have excess Fe (II)

Absorption saturation frequently occurs in X-ray absorption spectroscopy whenever transmission detection is used and the sample is too thick. This can strongly distort X-ray absorption spectra, particularly for transitions which have very high intensity and narrow line width relative to the instrumental resolution. This problem is particularly severe at the strong 2p → 3d resonances at the Ca 2p edge, such that there are detectible absorption saturation effects for mineral samples as thin as 30 nm. We describe methods to identify such spectral distortion in scanning transmission X-ray microscopy (STXM) and avoid its effects when generating reference spectra by selecting spatial regions with undistorted spectra. We also show that taking absorption saturation into account is critical when STXM is applied to systems with thick regions. We outline an approach which can provide accurate thickness maps even in the regions where the thickness is such the absorption is saturated at the resonance peaks. Environmental samples such as biofilms, soil samples, sediments, and precipitates are typically heterogeneous in both composition and thickness. They often contain regions thicker than the saturation threshold, yet one would still like to analyse such samples correctly and quantitatively. By excluding the peak energies where absorption saturation distortion occurs, we show it is possible to achieve a meaningful quantitative analysis. We demonstrate the advantages of this approach in the context of mapping two different CaCO 3 species biomineralized within a natural river biofilm.

The polarization dependence (linear dichroism) of the C 1s X-ray absorption spectrum of individual multi-walled carbon nanotubes (MWCNTs) is measured using scanning transmission X-ray microscopy. A very strong dichroic effect is found in the C 1s --> pi* transition, with almost complete disappearance of this transition when the electric-field (E)-vector is aligned parallel to high-quality (low-defect) MWCNTs and maximum intensity when the E-vector is orthogonal to the tube axis. In contrast, there is very little dichroism in the C 1s --> sigma* transitions. The origin of this polarization effect is explained. The magnitude of the polarization dependence is found to differ in MWCNTs synthesized by different methods (arc discharge versus chemical vapor deposition). This is ascribed to differences in densities of sp(2)-type defects. The potential for use of this signal to characterize defects in single-carbon-nanotube devices is discussed.

An important feature of microbial communities is the spatial heterogeneity of extracellular chemistry. Scanning transmission X-ray microscopy (STXM) was used to map the spatial distribution of iron speciesthroughout Pseudomonas aeruginosa biofilms to assess the influence of chemical heterogeneity on biomineralization. P. aeruginosa biofilms were treated with Fe(III)-amended media. Speciation and quantitative mapping using STXM image sequences in the Fe 2p(3/2) (L3) absorption edge region revealed both Fe(II) and Fe(III) in localized microenvironments. Fe(III) was mainly associated with cell surfaces, while small amounts of Fe(II) was found in the extracellular space. Biofilms were also characterized using C 1s edge STXM image sequences. Anaerobic growth assays and confocal microscopy revealed the inability of P. aeruginosa to directly reduce Fe(III), implicating indirect iron reduction mechanisms in the formation of fine-grained, multivalent minerals. These studies suggest that geochemical microenvironments found throughout microbial communities are even more complex than previously believed.

Picocyanobacteria were found to play an important role in calcite precipitation in oligotrophic lakes. In this study, investigations on the interface between cyanobacteria and attached biogenic calcite crystals have been performed to gain further insights into the mechanisms of nucleation of these precipitates. Ultramicrotomy, the conventional preparation technique of thin sections for Transmission Electron Microscopy (TEM) investigations, often fails when working on heterogeneous samples containing soft organic material and hard minerals. Thus, in this study the thin sections were prepared using Focused Ion Beam (FIB) milling. This approach is usually applied in material sciences but until recently was not very common in environmental research. Different analytical TEM methods like Electron Spectroscopic Imaging (ESI) and Electron Energy Loss Spectrometry (EELS) were used to test the suitability of FIB-milling for the preparation of organic/inorganic interface specimens. With this approach we were able to analyze both organic and the inorganic phases of the same sample. Elemental maps of the samples were also calculated. By analyzing the structure of the C K -absorption edge, the different bonding forms of the organic carbon cell and the inorganic carbon of the crystal could be clearly distinguished.

An approach for long-term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution by Atomic Force Microscopy (AFM) was developed in this study. Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response to environmental changes and mineral nucleation at the cell's surface. Continuously cultured cyanobacteria of the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immobilized on poly-l-lysine-coated glass slides. Both immobilization and environmental conditions were optimized in order to facilitate long-term experiments (> 100 h) with living cells. AFM samples were investigated in situ in two different solutions: Culture medium was used for cultivation experiments and nutrient-free NaHCO3/CaCl2 solutions (supersaturated with respect to calcite) for long-term characterizations of the changes in cell surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence microscopy, independently. No indications for extended starvation were found within the relevant timescales. Analysing the influence of Ca2+ on the surface of S. leopoliensis, we found significant changes compared to a Ca-free solution. Few hours after CaCl2 was added to the circumfluent solution, small protuberances were observed on the cell surface. These are promising results to environmental scientists for a wide range of applications, as cell response to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit from this new approach.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.

The identification of environmental processes and mechanisms often requires information on the organochemical and inorganic composition of specimens at high spatial resolution. X-ray spectroscopy (XAS) performed in the soft X-ray range (100-2,200 eV) provides chemical speciation information for elements that are of high biogeochemical relevance such as carbon, nitrogen, and oxygen but also includes transition metals such as iron, manganese, or nickel. Synchrotron-based scanning transmission X-ray microscopy (STXM) combines XAS with high resolution mapping on the 20-nm scale. This provides two-dimensional (2D) quantitative information about the distribution of chemical species such as organic macromolecules, metals, or mineral phases within environmental samples. Furthermore, the combination of STXM with angle-scan tomography allows for three-dimensional (3D) spectromicroscopic analysis of bio-, geo-, or environmental samples. For the acquisition of STXM tomography data, the sample is rotated around an axis perpendicular to the X-ray beam. Various sample preparation approaches such as stripes cut from TEM grids or the preparation of wet cells allow for preparing environmentally relevant specimens in a dry or in a fully hydrated state for 2D and 3D STXM measurements. In this chapter we give a short overview about the principles of STXM, its application to environmental sciences, different preparation techniques, and the analysis and 3D reconstruction of STXM tomography data.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe 3+ , Cu 2+ , Zn 2+ , and Hg 2+ , illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

The alignment of β-sheets within spider dragline silk fibers is an important factor in their tensile strength and extensibility. We are using linear dichroism of the C 1s → π*amide transition measured using scanning transmission X-ray microscopy (STXM) to generate quantitative maps of the orientation parameters with 30 nm spatial resolution. Here we have extended these measurements from dry samples to samples with partial or full hydration. A device for monitoring and controlling the humidity of a sample in the STXM is described and used to measure the effect of saturated humidity on a section of N. clavipes dragline spider silk. The microstructure and distributions of molecular orientation change considerably with hydration in ways consistent with the supercontraction observed in free standing dragline spider silk. The STXM results are compared to infrared and Raman microscopy results.