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.

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.

Precise 3D image alignment in micro-axial tomography.

Micro (micro-) axial tomography is a challenging technique in microscopy which improves quantitative imaging especially in cytogenetic applications by means of defined sample rotation under the microscope objective. The advantage of micro-axial tomography is an effective improvement of the precision of distance measurements between point-like objects. Under certain circumstances, the effective (3D) resolution can be improved by optimized acquisition depending on subsequent, multi-perspective image recording of the same objects followed by reconstruction methods. This requires, however, a very precise alignment of the tilted views. We present a novel feature-based image alignment method with a precision better than the full width at half maximum of the point spread function. The features are the positions (centres of gravity) of all fluorescent objects observed in the images (e.g. cell nuclei, fluorescent signals inside cell nuclei, fluorescent beads, etc.). Thus, real alignment precision depends on the localization precision of these objects. The method automatically determines the corresponding objects in subsequently tilted perspectives using a weighted bipartite graph. The optimum transformation function is computed in a least squares manner based on the coordinates of the centres of gravity of the matched objects. The theoretically feasible precision of the method was calculated using computer-generated data and confirmed by tests on real image series obtained from data sets of 200 nm fluorescent nano-particles. The advantages of the proposed algorithm are its speed and accuracy, which means that if enough objects are included, the real alignment precision is better than the axial localization precision of a single object. The alignment precision can be assessed directly from the algorithm's output. Thus, the method can be applied not only for image alignment and object matching in tilted view series in order to reconstruct (3D) images, but also to validate the experimental performance (e.g. mechanical precision of the tilting). In practice, the key application of the method is an improvement of the effective spatial (3D) resolution, because the well-known spatial anisotropy in light microscopy can be overcome. This allows more precise distance measurements between point-like objects.