Florian Staier

Coherent imaging of biological samples with femtosecond pulses at the free-electron laser FLASH

Coherent x-ray imaging represents a new window to imaging non- crystalline, biological specimens at unprecedented resolutions. The advent of free-electron lasers (FEL) allows extremely high flux densities to be delivered to a specimen resulting in stronger scattered signal from these samples to be measured. In the best case scenario, the diffraction pattern is measured before the sample is destroyed by these intense pulses, as the processes involved in radiation damage may be substantially slower than the pulse duration. In this case, the scattered signal can be interpreted and reconstructed to yield a faithful image of the sample at a resolution beyond the conventional radiation damage limit. We employ coherent x-ray diffraction imaging (CXDI) using the free-electron

Precise 3D image alignment in micro-axial tomography

Micro (µ‐) 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 µ‐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 algorithms 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.

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.

Digital in-line soft x-ray holography with element contrast

Digital in-line soft x-ray holography (DIXH) was used to image immobilized polystyrene and iron oxide particles and to distinguish them based on their different x-ray absorption cross sections in the vicinity of the carbon K-absorption edge. The element-specific information from the resonant DIXH images was correlated with high-resolution scanning electron microscopy (SEM) pictures. We also present DIXH images of a cell nucleus and compare the contrast obtained for nuclear components with the appearance in optical microscopy.

Digital In-line Holography with femtosecond VUV radiation provided by the free-electron laser FLASH

Femtosecond vacuum ultraviolet (VUV) radiation provided by the free-electron laser FLASH was used for digital in-line holographic microscopy and applied to image particles, diatoms and critical point dried fibroblast cells. To realize the classical in-line Gabor geometry, a 1 microm pinhole was used as spatial filter to generate a divergent light cone with excellent pointing stability. At a fundamental wavelength of 8 nm test objects such as particles and diatoms were imaged at a spatial resolution of 620 nm. In order to demonstrate the applicability to biologically relevant systems, critical point dried rat embryonic fibroblast cells were for the first time imaged with free-electron laser radiation.

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.

Soft X-ray holographic microscopy of chromosomes with high aspect ratio pinholes

We used digital in-line soft X-ray holography (DIXH) in the Gabor geometry to image human chromosomes. The divergent wave front was generated by diffraction of synchrotron radiation from a high aspect ratio pinhole. As under our experimental conditions the achievable resolution depends on the pinhole radius, high aspect ratio holes with diameters in the 100 nm range were prepared by focused ion beam (FIB) milling. The central maximum of the obtained Airy pattern was used to image chromosomes prepared from metaphase HeLa cells at experimental resolutions of 370±40 nm with soft X-rays at 260 eV photon energy provided by the BESSY II synchrotron radiation facility.

Micro axial tomography: A miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

Reconstruction of high-resolution fluorescence microscopy images based on axial tomography

For a reliable understanding of cellular processes, high resolution 3D images of the investigated cells are necessary. Unfortunately, the ability of fluorescence microscopes to image a cell in 3D is limited since the resolution along the optical axis is by a factor of two to three worse than the transversal resolution. Standard microscopy image deblurring algorithms like the Total Variation regularized Richardson Lucy algorithm are able to improve the resolution but the problem of a lower resolution in direction along the optical axis remains. However, it is possible to overcome this problem using Axial Tomography providing tilted views of the object by rotating it under the microscope. The rotated images contain additional information about the objects and an advanced method to reconstruct a 3D image with an isotropic resolution is presented here. First, bleaching has to be corrected in order to allow a valid registration correcting translational and rotational shifts. Hereby, a multi-resolution rigid registration method is used in our method. A single high-resolution image can be reconstructed on basis of all aligned images using an extended Richardson Lucy method. In addition, a Total Variation regularization is applied in order to guarantee a stable reconstruction result. The results for both simulated and real data show a considerable improvement of the resolution in direction of the optical axis.