Cryogenic X-ray diffraction microscopy for biological samples.

X-ray diffraction microscopy (XDM) is well suited for nondestructive, high-resolution biological imaging, especially for thick samples, with the high penetration power of x rays and without limitations imposed by a lens. We developed nonvacuum, cryogenic (cryo-) XDM with hard x rays at 8 keV and report the first frozen-hydrated imaging by XDM. By preserving samples in amorphous ice, the risk of artifacts associated with dehydration or chemical fixation is avoided, ensuring the imaging condition closest to their natural state. The reconstruction shows internal structures of intact D. radiodurans bacteria in their natural contrast.

Surface roughness of stainless-steel mirrors for focusing soft x rays.

We have used polished stainless steel as a mirror substrate to provide focusing of soft x rays in grazing-incidence reflection. The critical issue of the quality of the steel surface, polished and coated with gold, is discussed in detail. A comparison is made to a polished, gold-coated, electroless nickel surface, which provides a smoother finish. We used the surface height distributions, measured with an interferometric microscope and complemented by atomic-force microscope measurements, to compute power spectral densities and then to evaluate the surface roughness. The effects of roughness in reducing the specular reflectivity were verified by soft-x-ray measurements.

High-resolution ab initio three-dimensional x-ray diffraction microscopy.

Coherent x-ray diffraction microscopy is a method of imaging nonperiodic isolated objects at resolutions limited, in principle, by only the wavelength and largest scattering angles recorded. We demonstrate x-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the three-dimensional diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a nonperiodic object. We also construct two-dimensional images of thick objects with greatly increased depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution with x-ray undulator radiation and establishes the techniques to be used in atomic-resolution ultrafast imaging at x-ray free-electron laser sources.

Biological imaging by soft x-ray diffraction microscopy.

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.

Suite of three protein crystallography beamlines with single superconducting bend magnet as the source.

At the Advanced Light Source, three protein crystallography beamlines have been built that use as a source one of the three 6 T single-pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single-pole superconducting bend magnets enables the development of a hard X-ray program on a relatively low-energy 1.9 GeV ring without taking up insertion-device straight sections. The source is of relatively low power but, owing to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double-crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

SPEDEN: reconstructing single particles from their diffraction patterns.

SPEDEN is a computer program that reconstructs the electron density of single particles from their X-ray diffraction patterns, using a single-particle adaptation of the holographic method in crystallography [Szöke, Szöke & Somoza (1997). Acta Cryst. A53, 291-313]. The method, like its parent, is unique because it does not rely on 'back' transformation from the diffraction pattern into real space and on interpolation within measured data. It is designed to deal successfully with sparse, irregular, incomplete and noisy data. It is also designed to use prior information for ensuring sensible results and for reliable convergence. This article describes the theoretical basis for the reconstruction algorithm, its implementation, and quantitative results of tests on synthetic and experimentally obtained data. The program could be used for determining the structures of radiation-tolerant samples and, eventually, of large biological molecular structures without the need for crystallization.