André Beerlink

Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·105 Gy, and were shown to scale with photon fluence.

Phase-contrast x-ray imaging and tomography of the nematode Caenorhabditis elegans

We have analyzed the model organism Caenorhabditis elegans with the help of phase-contrast x-ray tomography. This work combines techniques from x-ray imaging studies of single biological cells by in-line holography with three-dimensional reconstruction and furthermore extends these studies to the multicellular level. To preserve the sub-cellular ultrastructure of the nematodes, we used the near-native sample preparation of high-pressure freezing as commonly used in the field of electron microscopy. For the presented samples, a standard, non-magnifying parallel-beam setting, as well as a magnifying, divergent-beam setting using nanofocusing optics is evaluated based on their tomographic reconstruction potential. In this paper, we address difficulties in sample preparation and issues of image processing. By experimental refinement and through optimized reconstruction procedures, we were able to perform x-ray imaging studies on a living specimen.

Hard x-ray phase contrast imaging of black lipid membranes

We report hard x-ray phase contrast imaging of black lipid membranes, freely suspended over a micromachined aperture in an aqueous solution. Biomolecular and organic substances can thus be probed in hydrated environments by parallel beam propagation imaging, using coherent multi-kilo-electronvolt x-ray radiation. The width of the thinning film can be resolved from analysis of the intensity fringes in the Fresnel diffraction regime down to about 200 nm. The thinning process, in which solvent is expelled from the space in between two opposing monolayers, is monitored, and the domain walls between coexisting domains of swollen and thinned membrane patches are characterized.

X-ray Structure Analysis of Free-Standing Lipid Membranes Facilitated by Micromachined Apertures

Silicon and Teflon substrates have been structured by wet etching and a focused ion beam (FIB) to obtain very defined, clean apertures. Planar, free-standing lipid membranes (black lipid membranes (BLM)) with enhanced long-term stability have been prepared on these apertures by the methods of Montal and Müller(1,2) as well as Müller and Rudin.(3) The stability and geometric control enables the use of X-ray analysis of free-standing single bilayers. With the presented setup, simultaneous structural and electrophysiological measurements will become feasible.

X-Ray propagation imaging of a lipid bilayer in solution

We have used X-ray propagation imaging to visualize a less than 5 nm thick native lipid bilayer membrane freely suspended in aqueous solution. Contrast is formed by free space propagation of hard X-rays, with the membrane illuminated by a nano-focused, partially coherent synchrotron beam, at a controllable distance (defocus) behind the focal spot. Quantitative fitting of the magnified Fresnel fringes shows the transition from membranes swollen with solvent to the native bilayer, containing structural information at near-molecular resolution along the dimension perpendicular to the bilayer. We show first applications of this hybrid technique of propagation imaging and near-field diffraction to the investigation of ultra-thin organic films formed in micro-fluidic devices, namely the formation of a lipid bilayer by the adhesion of two constitutive monolayers.

Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering.

Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer.

A novel heavy-atom label for side-specific peptide iodination: synthesis, membrane incorporation and X-ray reflectivity.

Structural parameters, such as conformation, orientation and penetration depth of membrane-bound peptides and proteins that may function as channels, pores or biocatalysts, are of persistent interest and have to be probed in the native fluid state of a membrane. X-ray scattering in combination with heavy-atom labeling is a powerful and highly appropriate method to reveal the position of a certain amino acid residue within a lipid bilayer with respect to the membrane normal axis up to a resolution of several Angstrøm. Herein, we report the synthesis of a new iodine-labeled amino acid building block. This building block is intended for peptide incorporation to provide high intensities for electron density difference analysis of X-ray reflectivity data and improve the labeling potential for the lipid bilayer head-group and water region. The novel building block as well as the commercially available non-iodinated analogue, required for X-ray scattering, was implemented in a transmembrane peptide motif via manual solid-phase peptide synthesis (SPPS) following the fluorenylmethyloxycarbonyl (Fmoc)-strategy. The derived peptides were reconstituted in lipid vesicles as well as in highly aligned multilamellar lipid stacks and investigated via circular dichroism (CD) and X-ray reflectivity. Thereby, it has been revealed that the bulky iodine probe neither causes conformational change of the peptide structure nor lamellar disordering of the membrane complexes.

Improvement of SAXS Lab Equipment by Using Scatterless Apertures

Parasitic scattering caused by apertures is a well-known problem in X-ray analytics, which forces users and manufacturers to adapt their experimental setup to this unwanted phenomenon. Increased measurement times due to lower photon fluxes, a lower resolution caused by an enlarged beam stop, a larger beam defining pinhole-to-sample distance due to the integration of an antiscatter guard and generally a lower signal-to-noise ratio leads to a loss in data quality. In this presentation we will explain how the lately developed scatterless pinholes called SCATEX overcome the aforementioned problems. SCATEX pinholes are either made of Germanium or of Tantalum and momentarily have a minimum diameter of 30μm. Thus, these novel apertures are applicable to a wide range of different applications and X-ray energies. We will show measurements which were performed either at home-lab small angle X-ray scattering (SAXS) systems such as the NANOSTAR of Bruker AXS or at synchrotron beamlines. At the PTB four-crystal monochromator beamline at BESSY II data was collected for a comparison of conventional pinholes, scatterless Germanium slit systems and SCATEX pinholes. At the Nanofocus Endstation P03 beamline at PETRA III we compared the performance of our SCATEX apertures with conventional Tungsten slit systems under high flux density conditions.

Upgrading Existing Diffractometers with State-of-the-art Microfocus Sources

Modern microfocus X-ray sources define the state-of-the-art for a broad spectrum of applications in home laboratories, such as protein and small molecule crystallography, and small-angle scattering. These sources are combined with multilayer optics to image the source spot onto the sample. The optics provides a parallel or focused monochromatic X-ray beam, magnified to a suitable size. Low power sealed microfocus sources, such as Incoatec’s IμS represent an attractive alternative to rotating anodes, with a significant reduction in cost and maintenance. Power loads of a few kW/mm2 in anode spot sizes below 50μm deliver a compact brilliant beam. For example, the IμSHighBrilliance delivers up to 1010 photons/s/mm2 in a spot size in the 100μm range. It is available for Cu, Mo, Ag, Cr and Co anodes. Since the launch in 2006 more than 400 IμS are now in operation worldwide for a large variety of applications in biology, chemistry, physics and material science. Are you tired of getting spare parts for an ancient rotating anode or is your detector performance only limited by your beam delivery system? We will demonstrate how to bring former high end diffractometers back to a superb performance for cutting edge science after an upgrade with an IμS source. Incoatec ensures full software and safety integration, and an installation hand in hand with the local service, providing a constant service support from your partners on site. In addition to all Bruker or Nonius systems, Incoatec also offers integrations into a wide range of instruments from Rigaku, Marresearch or STOE, also with Dectris or Huber components.

Hard X‐Ray Phase Contrast Imaging of Black Lipid Membranes

We image black lipid membranes based on hard x‐ray Fresnel diffraction, with phase contrast arising from free space propagation of the beam traversing the sample. We show that for the studied membranes a simplified but extendable model can be used to quantitatively monitor the thickness and its changes during the thinning process of BLMs.