B. Niemann

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.

PHASE CONTRAST STUDIES OF BIOLOGICAL SPECIMENS WITH THE X-RAY MICROSCOPE AT BESSY (INVITED)

The transmission x‐ray microscope at the BESSY storage ring is being used for investigations in the fields of biology, biophysics, medicine, colloid chemistry, and soil sciences. The system has been instrumented for phase contrast studies with the possibility of quick change between imaging in amplitude and phase contrast mode. Phase contrast has the advantage of considerable higher image contrast and reduced radiation dosage compared to amplitude contrast. A cryogenic object chamber has been implemented on the x‐ray microscope. First experiments have shown that at cryogenic temperatures the structural stability of biological specimens is increased by three orders of magnitude in comparison with unfixed wet specimens at room temperature.

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.

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)

X‐RAY MICROSCOPY OF BIOLOGICAL SPECIMENS WITH A ZONE PLATE MICROSCOPE

X-ray microscopy, with wavelengths of a few tenths of a nanometer generated by conventional x-ray sources, has been used for several decades. Besides the technique of contact microradiography, various methods have been developed to obtain magnified images with x-rays by using grazing incidence mirror optics or projection techniques. This classical work, done mainly for biological and partly for medical and physical, (e.g., metallographic) research, has been described in many reviews.-5 The resolution achieved was not significantly better than that of a light microscope. The reasons for this were the lack of high resolution optical elements, the great penetration of the hard x-rays used, and the weakness of the classical x-ray sources. During the last several years, the situation has changed substantially. First, synchrotron radiation sources now give a high flux in the wavelength range from 1 to 10 nm. Second, methods to make x-ray lenses for this wavelength range have developed rapidly. Finally, the technique of contact microradiography has progressed considerably through the use of high resolution photosensitive resist materials in combination with scanning electron microscopes. The wavelength range 1-10 nm is best suited for high resolution x-ray microscopy of biological specimens, mainly because of the higher total absorption cross sections, the absence of scattering, and the low photon energy in comparison to the harder x-ray photons. This latter fact has already been stressed by several and has been theoretically investigated by Sayre et al. lop when they compared radiation dosages in electron and soft x-ray microscopy of biological materials in a natural state. Synchrotron radiation was not used for soft x-ray microscopy before 1972, when Horowitz and Howell demonstrated the feasibility of scanning microscopy, though with a resolution of only 2 pm. In 1976, Niemann el aL13 made enlarged

Tomographic imaging of biological specimens with the cryo transmission X-ray microscope

Using the photoelectric absorption contrast between water and protein at 2:4 nm wavelength, cryo X-ray microscopy has visualized protein structures down to 30 nm size in unstained, unsectioned biological specimens. Due to the large depth of focus of the Fresnel zone plate objectives, computed tomography based on a tilt series of X-ray microscopic images can be used to reconstruct the three-dimensional specimen structure. This method has been applied to the green alga Chlamydomonas reinhardtii, and to cell nuclei of male Drosophila melanogaster fruit fly cells. # 2001 Elsevier Science B.V. All rights reserved. PACS: 07.85.Tt; 87.59.Fm; 07.20.Mc