Matthias Bartels

Hard X-ray imaging of bacterial cells: nano-diffraction and ptychographic reconstruction.

Ptychographic coherent X-ray diffractive imaging (PCDI) has been combined with nano-focus X-ray diffraction to study the structure and density distribution of unstained and unsliced bacterial cells, using a hard X-ray beam of 6.2keV photon energy, focused to about 90nm by a Fresnel zone plate lens. While PCDI provides images of the bacteria with quantitative contrast in real space with a resolution well below the beam size at the sample, spatially resolved small angle X-ray scattering using the same Fresnel zone plate (cellular nano-diffraction) provides structural information at highest resolution in reciprocal space up to 2nm(-1). We show how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup. In addition, we present 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography.

Sub-5 nm hard x-ray point focusing by a combined Kirkpatrick-Baez mirror and multilayer zone plate

Compound optics such as lens systems can overcome the limitations concerning resolution, efficiency, or aberrations which fabrication constraints would impose on any single optical element. In this work we demonstrate unprecedented sub-5 nm point focusing of hard x-rays, based on the combination of a high gain Kirkpatrick-Baez (KB) mirror system and a high resolution W/Si multilayer zone plate (MZP) for ultra-short focal length f. The pre-focusing allows limiting the MZP radius to below 2 μm, compatible with the required 5 nm structure width and essentially unlimited aspect ratios, provided by enabling fabrication technology based on pulsed laser deposition (PLD) and focused ion beam (FIB).

Optogenetic stimulation of the auditory pathway

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.

Three-dimensional coordinates of individual atoms in materials revealed by electron tomography

Crystallography, the primary method for determining the 3D atomic positions in crystals, has been fundamental to the development of many fields of science. However, the atomic positions obtained from crystallography represent a global average of many unit cells in a crystal. Here, we report, for the first time, the determination of the 3D coordinates of thousands of individual atoms and a point defect in a material by electron tomography with a precision of ∼19 pm, where the crystallinity of the material is not assumed. From the coordinates of these individual atoms, we measure the atomic displacement field and the full strain tensor with a 3D resolution of ∼1 nm(3) and a precision of ∼10(-3), which are further verified by density functional theory calculations and molecular dynamics simulations. The ability to precisely localize the 3D coordinates of individual atoms in materials without assuming crystallinity is expected to find important applications in materials science, nanoscience, physics, chemistry and biology.

Sub-10 nm beam confinement by X-ray waveguides: design, fabrication and characterization of optical properties

Optimized X-ray waveguides have been fabricated and characterized in terms of transmission, angular acceptance, farfield pattern and imaging applications. Beam confinement down to sub-10 nm in two orthogonal directions has been demonstrated, at the nano-focus endstation at P10 of PETRA III at HASYLAB/DESY.

Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs

We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium.

Phase contrast tomography of the mouse cochlea at microfocus x-ray sources

We present phase contrast x-ray tomography of functional soft tissue within the bony cochlear capsule of mice, carried out at laboratory microfocus sources with well-matched source, detector, geometry, and reconstruction algorithms at spatial resolutions down to 2 μm. Contrast, data quality and resolution enable the visualization of thin membranes and nerve fibers as well as automated segmentation of surrounding bone. By complementing synchrotron radiation imaging techniques, a broad range of biomedical applications becomes possible as demonstrated for optogenetic cochlear implant research.

Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction

A compound optical system for coherent focusing and imaging at the nanoscale is reported, realised by high-gain fixed-curvature elliptical mirrors in combination with X-ray waveguide optics or different cleaning apertures. The key optical concepts are illustrated, as implemented at the Göttingen Instrument for Nano-Imaging with X-rays (GINIX), installed at the P10 coherence beamline of the PETRA III storage ring at DESY, Hamburg, and examples for typical applications in biological imaging are given. Characteristic beam configurations with the recently achieved values are also described, meeting the different requirements of the applications, such as spot size, coherence or bandwidth. The emphasis of this work is on the different beam shaping, filtering and characterization methods.

Transport of intensity phase reconstruction to solve the twin image problem in holographic x-ray imaging

We have implemented a deterministic method for solving the phase problem in hard x-ray in-line holography which overcomes the twin image problem. The phase distribution in the detector plane is retrieved by using two images with slightly different Fresnel numbers. We then use measured intensities and reconstructed phases in the detection plane to compute the exit wave in the sample plane. No further a priori information like a limited support or the assumption of pure phase objects is necessary so that it can be used for a wide range of complex samples. Using a nano-focused hard x-ray beam half period resolutions better than 30 nm are achieved.

Low-dose three-dimensional hard x-ray imaging of bacterial cells

We have imaged the three-dimensional density distribution of unstained and unsliced, freeze-dried cells of the gram-positive bacterium Deinococcus radiodurans by tomographic x-ray propagation microscopy, i.e. projection tomography with phase contrast formation by free space propagation. The work extends previous x-ray imaging of biological cells in the simple in-line holography geometry to full three-dimensional reconstruction, based on a fast iterative phase reconstruction algorithm which circumvents the usual twin-image problem. The sample is illuminated by the highly curved wave fronts emitted from a virtual quasi-point source with 10 nm cross section, realized by two crossed x-ray waveguides. The experimental scheme allows for a particularly dose efficient determination of the 3D density distribution in the cellular structure.