Single-Slice XRF Mapping of Light Elements in Frozen-Hydrated Allium schoenoprasum via a Self-Absorption-Corrected Hyperspectral Tomographic Reconstruction Approach.

3D and 2D-cross-sectional X-ray fluorescence analysis of biological material is a powerful tool to image the distribution of elements and to understand and quantify metal homeostasis and the distribution of anthropogenic metals and nanoparticles with minimal preparation artifacts. Using tomograms recorded on cryogenically prepared leaves of Allium schoenoprasum, the cross-sectional distribution of physiologically relevant elements like calcium, potassium, manganese, and zinc could be tomographically reconstructed by peak fitting followed by a conventional maximum-likelihood algorithm with self-absorption correction to reveal the quantitative cross-sectional element distribution. If light elements such as S and P are located deep in the sample compared to the escape depth of their characteristic X-ray fluorescence lines, the quantitative reconstruction becomes inaccurate. As a consequence, noise is amplified to a magnitude where it might be misinterpreted as actual concentration. We show that a tomographic MCA hyperspectral reconstruction in combination with a self-absorption correction allows for fitting of the XRF spectra directly in real space, which significantly improves the qualitative and quantitative analysis of the light elements compared to the conventional method as noise and artifacts in the tomographic reconstruction are reduced. This reconstruction approach can substantially improve the quantitative analysis of trace elements as it allows the fitting of summed voxel spectra in anatomical regions of interest. The presented method can be applied to XRF 2D single-slice tomography data and 3D tomograms and is particularly relevant for, but not limited to, biological material in order to help retrieve self-absorption corrected quantitative reconstructions of the spatial distribution of light elements and ultra-trace-elements.

X-ray fluorescence analysis of metal distributions in cryogenic biological samples using large-acceptance-angle SDD detection and continuous scanning at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III.

A new Rococo 2 X-ray fluorescence detector was implemented into the cryogenic sample environment at the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III, DESY, Hamburg, Germany. A four sensor-field cloverleaf design is optimized for the investigation of planar samples and operates in a backscattering geometry resulting in a large solid angle of up to 1.1 steradian. The detector, coupled with the Xspress 3 pulse processor, enables measurements at high count rates of up to 106 counts per second per sensor. The measured energy resolution of ∼129 eV (Mn Kα at 10000 counts s-1) is only minimally impaired at the highest count rates. The resulting high detection sensitivity allows for an accurate determination of trace element distributions such as in thin frozen hydrated biological specimens. First proof-of-principle measurements using continuous-movement 2D scans of frozen hydrated HeLa cells as a model system are reported to demonstrate the potential of the new detection system.

Use of small-angle X-ray scattering to resolve intracellular structure changes of Escherichia coli cells induced by antibiotic treatment.

The application of small-angle X-ray scattering (SAXS) to whole Escherichia coli cells is challenging owing to the variety of internal constituents. To resolve their contributions, the outer shape was captured by ultra-small-angle X-ray scattering and combined with the internal structure resolved by SAXS. Building on these data, a model for the major structural components of E. coli was developed. It was possible to deduce information on the occupied volume, occurrence and average size of the most important intracellular constituents: ribosomes, DNA and proteins. E. coli was studied after treatment with three different antibiotic agents (chloramphenicol, tetracycline and rifampicin) and the impact on the intracellular constituents was monitored.

Attachment of Algal Cells to Zwitterionic Self-Assembled Monolayers Comprised of Different Anionic Compounds.

The influence of zwitterionic self-assembled monolayers on settlement and removal of algae was studied. The monolayers were constructed either from zwitterionic thiols or from solutions of positively and negatively charged thiols. The cationic component was composed of quaternary ammonium terminated thiols and the anionic component contained sulfate or carboxylate termination. During assembly, all surfaces showed a strong tendency for equilibration of the surface charge. Settlement and adhesion assays with zoospores of Ulva linza and the diatom Navicula incerta, and field tests of the initial surface colonization revealed the relevance of charge equilibration for the biological inertness of the prepared surfaces.

Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action.

Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

Microfluidic detachment assay to probe the adhesion strength of diatoms.

Fouling release (FR) coatings are increasingly applied as an environmentally benign alternative for controlling marine biofouling. As the technology relies on removing fouling by water currents created by the motion of ships, weakening of adhesion of adherent organisms is the key design goal for improved coatings. In this paper, a microfluidic shear force assay is used to quantify how easily diatoms can be removed from surfaces. The experimental setup and the optimization of the experimental parameters to study the adhesion of the diatom Navicula perminuta are described. As examples of how varying the physico-chemical surface properties affects the ability of diatoms to bind to surfaces, a range of hydrophilic and hydrophobic self-assembled monolayers was compared. While the number of cells that attached (adhered) was barely affected by the coatings, the critical shear stress required for their removal from the surface varied significantly.

Conditioning of self-assembled monolayers at two static immersion test sites along the east coast of Florida and its effect on early fouling development.

Among the first events after immersion of surfaces in the ocean is surface 'conditioning'. Here, the accumulation and composition of the conditioning films formed after immersion in the ocean are analyzed. In order to account for different surface chemistries, five self-assembled monolayers that differ in resistance to microfouling and wettability were used. Water samples from two static immersion test sites along the east coast of Florida were collected at two different times of the year and used for experiments. Spectral ellipsometry revealed that conditioning films were formed within the first 24 h and contact angle goniometry showed that these films changed the wettability and rendered hydrophobic surfaces more hydrophilic and vice versa. Infrared reflection adsorption spectroscopy showed that the composition of the conditioning film depended on both the wettability and immersion site. Laboratory and field assays showed that the presence of a conditioning film did not markedly influence settlement of microorganisms.

Single pulse coherence measurements in the water window at the free-electron laser FLASH.

The spatial coherence of free-electron laser radiation in the water window spectral range was studied, using the third harmonic (λ<(3rd) = 2.66 nm) of DESY's Free-electron LASer in Hamburg (FLASH). Coherent single pulse diffraction patterns of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilamellar lipid stacks have been recorded. The intensity histogram of the speckle pattern around the first lamellar Bragg peak, corresponding to the d = 5 nm periodicity of the stack, reveals an average number of transverse modes of M¯ = 3.0 of the 3rd harmonic. Using the lipid stack as a 'monochromator', pulse-to-pulse fluctuations in the third harmonic λ(3rd) have been determined to be 0.033 nm.

Spatial and temporal coherence properties of single free-electron laser pulses.

The experimental characterization of the spatial and temporal coherence properties of the free-electron laser in Hamburg (FLASH) at a wavelength of 8.0 nm is presented. Double pinhole diffraction patterns of single femtosecond pulses focused to a size of about 10×10 µm(2) were measured. A transverse coherence length of 6.2 ± 0.9 µm in the horizontal and 8.7 ± 1.0 µm in the vertical direction was determined from the most coherent pulses. Using a split and delay unit the coherence time of the pulses produced in the same operation conditions of FLASH was measured to be 1.75 ± 0.01 fs. From our experiment we estimated the degeneracy parameter of the FLASH beam to be on the order of 10(10) to 10(11), which exceeds the values of this parameter at any other source in the same energy range by many orders of magnitude.

Surface sensing and settlement strategies of marine biofouling organisms.

This review article summarizes some recent insights into the strategies used by marine organisms to select surfaces for colonization. While larger organisms rely on their sensory machinery to select surfaces, smaller microorganisms developed less complex but still effective ways to probe interfaces. Two examples, zoospores of algae and barnacle larvae, are discussed and both appear to have build-in test mechanisms to distinguish surfaces with different physicochemical properties. Some systematic studies on the influence of surface cues on exploration, settlement and adhesion are summarized. The intriguing notion that surface colonization resembles a parallelized surface sensing event is discussed towards its complementarity with conventional surface analytical tools. The strategy to populate only selected surfaces seems advantageous as waves, currents and storms constantly challenge adherent soft and hard fouling organism.

Three dimensional tracking of exploratory behavior of barnacle cyprids using stereoscopy.

Surface exploration is a key step in the colonization of surfaces by sessile marine biofoulers. As many biofouling organisms can delay settlement until a suitable surface is encountered, colonization can comprise surface exploration and intermittent swimming. As such, the process is best followed in three dimensions. Here we present a low-cost transportable stereoscopic system consisting of two consumer camcorders. We apply this novel apparatus to behavioral analysis of barnacle larvae (≈800 µm length) during surface exploration and extract and analyze the three-dimensional patterns of movement. The resolution of the system and the accuracy of position determination are characterized. As a first practical result, three-dimensional swimming trajectories of the cypris larva of the barnacle Semibalanus balanoides are recorded in the vicinity of a glass surface and close to PEG2000-OH and C(11)NMe(3)(+)Cl(-) terminated self-assembled monolayers. Although less frequently used in biofouling experiments due to its short reproductive season, the selected model species [Marechal and Hellio (2011), Int Biodeterior Biodegrad, 65(1):92-101] has been used following a number of recent investigations on the settlement behavior on chemically different surfaces [Aldred et al. (2011), ACS Appl Mater Interfaces, 3(6):2085-2091]. Experiments were scheduled to match the availability of cyprids off the north east coast of England so that natural material could be used. In order to demonstrate the biological applicability of the system, analysis of parameters such as swimming direction, swimming velocity and swimming angle are performed.

Microfluidic assay to quantify the adhesion of marine bacteria.

For both, environmental and medical applications, the quantification of bacterial adhesion is of major importance to understand and support the development of new materials. For marine applications, the demand is driven by the quest for improved fouling-release coatings. To determine the attachment strength of bacteria to coatings, a microfluidic adhesion assay has been developed which allows probing at which critical wall shear stress bacteria are removed from the surface. Besides the experimental setup and the optimization of the assay, we measured adhesion of the marine bacterium Cobetia marina on a series of differently terminated self-assembled monolayers. The results showed that the adhesion strength of C. marina changes with surface chemistry. The difference in critical shear stress needed to remove bacteria can vary by more than one order of magnitude if a hydrophobic material is compared to an inert chemistry such as polyethylene glycol.

Settlement behavior of zoospores of Ulva linza during surface selection studied by digital holographic microscopy.

Settlement of the planktonic dispersal stages of marine organisms is the crucial step for the development of marine biofouling. Four-dimensional holographic tracking reveals the mechanism by which algal spores select surfaces suitable for colonization. Quantitative analysis of the three dimensional swimming trajectories of motile spores of a macroalga (Ulva linza) in the vicinity of surfaces functionalized with different chemistries reveals that their search strategy and swimming behavior is correlated to the number of settled spores found in spore settlement bioassays conducted over 45 min. The spore motility and exploration behavior can be classified into different motion patterns, with their relative occurrence changing with the surface chemistry. Based on the detailed motility analysis we derived a model for the surface selection and settlement process of Ulva zoospores.

Digital in-line X-ray holography with zone plates.

Single pulse imaging with radiation provided by free-electron laser sources is a promising approach towards X-ray microscopy, which is expected to provide high resolution images of biological samples unaffected by radiation damage. One fully coherent imaging technique for this purpose is digital in-line holography. Key to its successful application is the creation of X-ray point sources with high photon flux. In this study we applied zone plates to create such point sources with synchrotron radiation provided by the storage ring BESSY II. The obtained, divergent light cone is applied to holographic microscopy of biological objects such as critical point dried Navicula perminuta diatoms and human cells using photons with an energy of 250 eV. Compared to conventional experiments employing pinholes, exposure times are reduced by two orders of magnitude.

X-ray holographic microscopy with zone plates applied to biological samples in the water window using 3rd harmonic radiation from the free-electron laser FLASH.

The imaging of hydrated biological samples - especially in the energy window of 284-540 eV, where water does not obscure the signal of soft organic matter and biologically relevant elements - is of tremendous interest for life sciences. Free-electron lasers can provide highly intense and coherent pulses, which allow single pulse imaging to overcome resolution limits set by radiation damage. One current challenge is to match both the desired energy and the intensity of the light source. We present the first images of dehydrated biological material acquired with 3rd harmonic radiation from FLASH by digital in-line zone plate holography as one step towards the vision of imaging hydrated biological material with photons in the water window. We also demonstrate the first application of ultrathin molecular sheets as suitable substrates for future free-electron laser experiments with biological samples in the form of a rat fibroblast cell and marine biofouling bacteria Cobetia marina.

Ptychographic coherent x-ray diffractive imaging in the water window.

Coherent x-ray diffractive microscopy enables full reconstruction of the complex transmission function of an isolated object to diffraction-limited resolution without relying on any optical elements between the sample and detector. In combination with ptychography, also specimens of unlimited lateral extension can be imaged. Here we report on an application of ptychographic coherent diffractive imaging (PCDI) in the soft x-ray regime, more precisely in the so-called water window of photon energies where the high scattering contrast between carbon and oxygen is well-suited to image biological samples. In particular, we have reconstructed the complex sample transmission function of a fossil diatom at a photon energy of 517 eV. In imaging a lithographically fabricated test sample a resolution on the order of 50 nm (half-period length) has been achieved. Along with this proof-of-principle for PCDI at soft x-ray wavelengths, we discuss the experimental and technical challenges which can occur especially for soft x-ray PCDI.

Analysis of holographic microscopy data to quantitatively investigate three-dimensional settlement dynamics of algal zoospores in the vicinity of surfaces.

In this article we describe the technical aspects of digital in-line holographic microscopy to track multiple macrofouling Ulva linza zoospores simultaneously during their exploration of surfaces. Using an effective method of artefact suppression at the edges of holograms in combination with projection of volume reconstructions, a fast algorithm was developed which allows a reliable determination of a large number of subsequent spore positions. Thus, statistical analysis of swimming behaviour in the vicinity of surfaces becomes possible. Using glass surfaces as example, velocity and diving direction distributions are calculated and the swimming behaviour is statistically analysed. Diving direction analysis provides a straightforward way to determine segments within traces with surface contact. The presented method of data analysis allows high throughput analysis of holographic microscopy data and sets the basis for different applications including biofouling.

Coherent-pulse 2D crystallography using a free-electron laser x-ray source.

Coherent diffractive imaging for the reconstruction of a two-dimensional (2D) finite crystal structure with a single pulse train of free-electron laser radiation at 7.97 nm wavelength is demonstrated. This measurement shows an advance on traditional coherent imaging techniques by applying it to a periodic structure. It is also significant that this approach paves the way for the imaging of the class of specimens which readily form 2D, but not three-dimensional crystals. We show that the structure is reconstructed to the detected resolution, given an adequate signal-to-noise ratio.

Vacuum-ultraviolet Gabor holography with synchrotron radiation.

We present the realization of high-resolution holographic microscopy using the original Gabor geometry and imaging with radiation in the vacuum-ultraviolet (VUV) spectral region. Synchrotron VUV radiation with a wavelength of 13.8 nm was focused on a small pinhole generating a highly divergent light cone suitable for digital in-line holography. Objects of different thickness and materials have been used to test the imaging properties of holographic microscopy in the VUV wavelength range. The effective numerical aperture was limited by the illuminated area of the detector, yielding a theoretical resolution below 1 microm and an experimental one of approximately 1 microm.