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X-ray microscopy projects X-ray microscopy applications microspectroscopy and spectromicroscopy X-ray optics X-ray sources.

The condenser zone plate KZP7 is used not only in the Gottingen X-ray microscope at BESSY in Berlin, but also in the X-ray microscopes at ALS in Berkeley, USA, and at ISA in Aarhus, Danmark. The zone plate interference patten of KZP7 is generated with uv laser light using an optical system with two aspheric mirrors. The zone plate pattern is recorded in photoresist. To obtain optimum contrast an optically matched layer system is used. It reduces backreflection to the photoresist layer. The photoresist pattern is transferred into germanium by an trilevel reactive ion etching process.

It is planned, to use an undulator at BESSY II as source for a transmission X-ray microscope (TXM) and a scanning transmission X-ray microscope (STXM). The undulator with a magnetic period of 41 mm (U41) will be installed in a low β - section and will allow to cover the water window wavelength region in the first harmonic. The characteristics of this undulator will be presented.

A laboratory x-ray microscope has been built with zone plate optics and a pulsed plasma x-ray source. X-ray images of metal patterns, diatoms and 3T3 mouse cells have been taken with sub-optical resolution at a wavelength of about 2.5 nm.

The ability to interact with the environment is an essential feature of living biological systems and corresponds critically to their sensitivity to changes of environmental conditions. The investigation of biological objects and in particular preparation methods have to take into account this genuine lack of rigidity of living biological systems in order to avoid artefacts, i.e. unwanted changes of function or structure. In many cases x-ray microscopy is a well suited tool to investigate supramolecular structures of biological specimens in their natural state.

Phase zone plates with high efficiencies are required in x-ray microscopy to meet the demands of imaging specimens with a low radiation dose and a high contrast. Since the size of the resolution element is equal to the width of the outermost zone and the phase shift is a function of the thickness of the used zone plate material, the fabrication of phase zone plates can lead to high aspect ratios, i.e. to high ratios of the height to the width of the zone.

X-ray microscopy allows the investigation of the ultrastructure of biological samples in their native state with higher resolution than can be achieved with optical microscopy. We have used an X-ray microscope equipped with a special chamber for samples in aqueous solutions to study isolated polytene chromosomes in different states of condensation. Images of chromosomes were acquired using a special camera designed for the use of nuclear research plates with high detective quantum efficiency, thereby allowing shorter exposure times.

Micro zone plates with outermost zone widths of 40 nm, 35 nm and 30 nm have been used and characterized in the Gottingen x-ray microscope at BESSY. These were made through a collaboration between the IBM T.J. Watson Research Center and the Lawrence Berkeley Laboratory by electron beam writing. Condenser zone plates were made at the Forschungseinrichtung Rontgenphysik using a holographic method.

In x-ray microscopy experiments performed up to now the contrast is dominated by photoelectric absorption. The resulting radiation dose, though much less than in electron microscopy, limits fundamentally the resolution which can be obtained when investigating biological materials in the natural (intact wet and unstained) or living state, Sayre et al. (1977). Investigations to decouple resolution and radiation dose leads to a proposal for a phase contrast x-ray microscope. This work is based on experimental results on phase zone plates, Hilkenbach and Thieme (1986), and on the atomic scattering factors published by Henke (1981).

The limitations of the imaging techniques which dominate submicron biological imaging today, namely optical microscopy, diffraction of x-rays and electron microscopy, are due to the imaging particles themselves [1]. In optical microscopy the wavelength of the visible-light photon limits the resolution to about 0.2 μm. In x-ray diffraction the relatively low coherent-scattering cross-section of the hard x-ray photon makes it necessary to use macroscopic specimens to obtain sufficient interaction for imaging. In electron microscopy the large cross-section of the scattering electron makes it necessary to disassamble the cell. In addition, the weakness of the dependence of cross-section on atomic number makes staining and dehydration necessary.

X-ray microscopy with zone plates as x-ray imaging elements and synchrotron radiation was started by our group in 1976 at the Deutsches Elektronensynchrotron (DESY), Hamburg [20.1]. This prototype x-ray microscope was equipped with a grazing incidence grating monochromator, a condenser zone plate, and a micro-zone-plate. The grating was a 100 mm laminar grating with 600 1/mm holographically made in our optical laboratory [20.2]. The zone plates resulted from early holographic work using commercially available optics for zone plate construction [20.1,3,4]. The obtained resolution was 200 nm [20.5].

Soft x-ray microscopy provides higher resolution than visible light microscopy combined with the advantage of an inherent contrast mechanism. Unlike electron microscopy, x-ray microscopy can be used to investigate comparably thick objects of several μm in thickness. The reason of the natural contrast is, that the atomic scattering factors strongly depend on the different atomic species contained in the object under examination. Between 2.4nm and 4.4nm there is about an order of magnitude difference between the absorption coefficients for water and organic components. The natural contrast mechanism and the relative low radiation damage (Niemann et al. 1986) makes the x-ray microscope an useful tool in biology. The absorption is only one effect described by the complex atomic scattering factor, the second effect (the phase shift) is an even more valuable contrast mechanism (Schmahl and Rudolph, 1987).

Up to the end of 1986 it was always stated that to a good approximation soft x-ray photons participate in only one reaction with matter, i.e. absorption of the photon in an atom, because the atomic cross sections for inelastic and elastic scattering are 4 to 5 orders of magnitude smaller than the atomic cross section for photoelectric absorption. But this is only half the truth.

Zone plates are circular gratings with radially increasing line density. The imaging with zone plates of zone numbers n ≳ 100 obeys the same laws as imaging with thin refractive lenses. With r1= radius of the innermost zone (Fig.8.1), rn = radius of the n-th zone, n = zone number and m = number of diffracted order, the focal length of a zone plate is approximately given by fm =r1 2 λ−1 m−1. Because fm ∝1/λ, a zone plate has to be used with quasimonochromatic radiation with a bandwidth Δλ given by λ/Δλ ⋍ n◦m. The width of the outermost zone is drn = rn/(2n). The smallest distance of two point sources which can be resolved with a zone plate is — according to the Rayleigh criterion — given by d δ = 1,22 drn/m.

Fresnel zone plates which are used as imaging lenses for soft x-rays are required to have high resolution, high efficiency, and low distortions. In terms of practical realisation, this means smallest possible widths of the outer zones, high aspect ratios of the absorber rings, and a positional accuracy of the structures to a fraction of the outer zone width. Additionally, the absorptance of the membranes carrying the zone plate has to be minimized.

X-ray microscopy experiments using x-ray optical systems are performed with synchrotron radiation of electron storage rings. The highest resolution obtained up to now in such experiments is about 50 nm. The exposure time to take pictures with this resolution is a few seconds. X-ray optical elements are under development which allow to improve the resolution to about 10 nm. For such high resolution experiments synchrotron radiation sources will not be replaced by other devices in the foreseeable future.