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.

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.

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.

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.

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.

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.

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.

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.