Abstract: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. (4) were well satisfied in our experimental geometry. The maximum time delay introduced through the path length difference was τ max ≈ 0.6 fs and was smaller than the temporal coherence length τ c = (1.75 ± 0.0.01) fs measured by the split and delay unit (see below). Therefore, we could safely assume that in transverse coherence measurements |γ eff 12 (τ)| ≈ |γ eff 12 (0)| and α 12 (τ) ≈ α 12 (0). 36. An unconstrained fit yields a value of |γ eff 11 | ≈ 0.8 in both directions and provides slightly larger values for the transverse coherence length. We attribute this to inhomogenities in the transmission through the pinholes. 37. J. Chalupsky, J. Krzywinski, L. Juha, V. Hajkova, J. Cihelka, T. Burian, L. Vyain, J. Gaudin, A. Gleeson, M.Jurek, A. R. Khorsand, D. Klinger, H. Wabnitz, R. Sobierajski, M. Störmer, K. Tiedtke, and S. Toleikis, "Spot size characterization of focused non-Gaussian X-ray laser beams", Opt. Express 18, 27836 (2010). 38. We attribute this positional uncertainty to both, instabilities of the sample stages and beam positional jitter. 39. In our experiment the maximum of |γ 12 (τ)| did not reach unity but rather a value of 0.14. The reason for this is that the full beam was split in the middle and overlapped again meaning that parts of the center of the beam were overlapped with parts of the edge of the beam (see [27]

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The unique strengths of x-ray microscopy are high penetration depth and near-edge resonances that provide chemical information. We use ptychography, a coherent diffractive imaging technique that disposes of the requirement for isolated specimens, and demonstrate resonant imaging by exploiting resonances near the oxygen K edge to differentiate between two oxygen-containing materials. To highlight a biological system where resonant ptychography might be used for chemical mapping of unsliced cells, reconstructions of freeze-dried Deinococcus radiodurans cells at an energy of 517 eV are shown.

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.

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.

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Abstract: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. ©2011 Optical Society of America References and links1. C. A. Brau, "Free-electron lasers," Science 239(4844), 1115-1121 (1988). 2. J. Feldhaus, J. Arthur, and J. Hastings, "X-ray free-electron lasers," J. Phys. B: At. Mol. Opt. Phys. 38(9), S799-S819 (2005

A ptychographical coherent diffractive imaging experiment in the water window with focused soft X-rays at 500 eV is reported. An X-ray beam with high degree of coherence was selected for ptychography at the P04 beamline of PETRA III synchrotron radiation source. The beam coherence was measured with the newly developed non-redundant array method, and a coherence length of 4.1 mm and global degree of coherence of 35% at 100 mm exit slit opening in the vertical direction were determined. A pinhole, 2.6 mm in size, selected the coherent part of the beam that was used to obtain ptychographic reconstruction results of a lithographically manufactured test sample and a fossil diatom. The achieved resolution was 53 nm for the test sample and was only limited by the size of the detector. The diatom was imaged at a resolution better than 90 nm.