D. Rudolph

Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.

X-ray microscopy and spectromicroscopy

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

Soft X-ray imaging zone plates with large zone numbers for microscopic and spectroscopic applications

Abstract Holographically made zone plates are described. Imaging properties and spectrometric applications are discussed. Experimental results with soft X-radiation are given.

Zone-plate X-ray microscopy

Fresnel zone plates are the most commonly used optic in x-ray microscopes. Following a short discussion of historical developments, the properties of zone plates are outlined, along with the microscope systems that employ them. A number of applications of x-ray microscopes are then surveyed, including in biology, environmental science, and materials science.

Zone Plates for X-Ray Microscopy

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.

The Göttingen X-Ray Microscope and X-Ray Microscopy Experiments at the BESSY Storage Ring

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].

Status Of The Sputtered Sliced Zone Plates For X-Ray Microscopy

Procedures are described to make sputtered sliced zone plates. These systems are under development for a resolution range of 10 nm ≤ d ≤ 50 nm.

The Göttingen X-ray microscopes☆

Abstract An X-ray microscope (XM) is described. This microscope contains a zone plate linear monochromator as a condenser system and high resolution micro zone plate. With this microscope X-ray microscopy experiments have been performed using synchroton radiation at the electron storage ring ACO in Orsay/Paris. Images with 50 nm resolution have been made. Additionally, a scanning X-ray microscope (SXM) is described. This system is under construction and is projected for a resolution of 10–50 nm.

Amplitude and Phase Contrast in X-Ray Microscopy

When a material object is placed in the path of an electromagnetic wave, the latter undergoes a change in both its amplitude and its phase. In particular, calculations have been performed previously for two extremes(1) to obtain the contrast caused purely by the change in amplitude produced when an object is imaged by soft X rays (an amplitude object) or, alternatively, by the change in phase (a phase object) when imaged in a phase-contrast microscope with coherent illumination. These calculations show that phase contrast exceeded amplitude contrast. In fact, phase-contrast microradiography(2) and phase-contrast topography using x-ray interferometers(3) had already been suggested several years prior to the work reported here. To confirm these calculations, preliminary phase-contrast x-ray microscopy experiments were performed with the x-ray microscope at the BESSY electron storage ring.(4.5)

Thermally driven shape instabilities of Nb/Cu multilayer structures: instability of Nb/Cu multilayers

The disintegration of Nb layers in Nb/Cu multilayers during heat treatments was investigated by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), and X-ray microscopy (XRM). Spherically shaped Nb particles were observed after a heat treatment at 870 K for 3 h. The growth velocity of the Nb particles depends strongly on the initial interface roughness and the Nb layer thickness. The XRM investigations indicate a process of self-assembly, leading to a non-statistical distribution of the particles. Measurements of the superconducting properties of both annealed and non-annealed samples, confirm the microstructural observations.