Use of extended and prepared reference objects in experimental Fourier transform X-ray holography

The use of one or more gold nanoballs as reference objects for Fourier Transform holography (FTH) is analysed using experimental soft X-ray diffraction from objects consisting of separated clusters of these balls. The holograms are deconvoluted against ball reference objects to invert to images, in combination with a Wiener filter to control noise. A resolution of ~30nm, smaller than one ball, is obtained even if a large cluster of balls is used as the reference, giving the best resolution yet obtained by X-ray FTH. Methods of dealing with missing data due to a beamstop are discussed. Practical prepared objects which satisfy the FTH condition are suggested, and methods of forming them described.Comment: 7 pages, 2 figures, submitted to Applied Physics Letter

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. (Szoke, A., Szoke, H., and Somoza, J.R., 1997. Acta Cryst. A53, 291-313.) The method, like its parent, is unique that 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 structure of radiation tolerant samples and, eventually, of large biological molecular structures without the need for crystallization.Comment: 12 pages, 10 figure

Dose, exposure time, and resolution in Serial X-ray Crystallography

The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed Serial Crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available molecular and X-ray fluxes and molecular alignment. Orientation of the diffracting molecules is achieved by laser alignment. We evaluate the incident X-ray fluence (energy/area) required to obtain a given resolution from (1) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (2) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (3) the frequency cut off of the transfer function following iterative solution of the phase problem, and reconstruction of an electron density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate counting time and the number of proteins needed within the beam at any instant for a given resolution and X-ray flux. We confirm an inverse fourth power dependence of exposure time on resolution, with important implications for all coherent X-ray imaging. We find that multiple single-file protein beams will be needed for sub-nanometer resolution on current third generation synchrotrons, but not on fourth generation designs, where reconstruction of secondary protein structure at a resolution of 0.7 nm should be possible with short exposures.Comment: 19 pages, 7 figures, 1 tabl

X-ray image reconstruction from a diffraction pattern alone

A solution to the inversion problem of scattering would offer aberration-free diffraction-limited 3D images without the resolution and depth-of-field limitations of lens-based tomographic systems. Powerful algorithms are increasingly being used to act as lenses to form such images. Current image reconstruction methods, however, require the knowledge of the shape of the object and the low spatial frequencies unavoidably lost in experiments. Diffractive imaging has thus previously been used to increase the resolution of images obtained by other means. We demonstrate experimentally here a new inversion method, which reconstructs the image of the object without the need for any such prior knowledge.Comment: 5 pages, 3 figures, improved figures and captions, changed titl

Phasing diffuse scattering. Application of the SIR2002 algorithm to the non-crystallographic phase problem

A new phasing algorithm has been used to determine the phases of diffuse elastic X-ray scattering from a non-periodic array of gold balls of 50 nm diameter. Two-dimensional real-space images, showing the charge-density distribution of the balls, have been reconstructed at 50 nm resolution from transmission diffraction patterns recorded at 550 eV energy. The reconstructed image fits well with scanning electron microscope (SEM) image of the same sample. The algorithm, which uses only the density modification portion of the SIR2002 program, is compared with the results obtained via the Gerchberg-Saxton-Fienup HIO algorithm. In this way the relationship between density modification in crystallography and the HiO algorithm used in signal and image processing is elucidated.Comment: 7 pages, 12 figure

Coherent X-ray Diffractive Imaging; applications and limitations

The inversion of a diffraction pattern offers aberration-free diffraction-limited 3D images without the resolution and depth-of-field limitations of lens-based tomographic systems, the only limitation being radiation damage. We review our experimental results, discuss the fundamental limits of this technique and future plans.Comment: 7 pages, 8 figure

Progress in Three-Dimensional Coherent X-Ray Diffraction Imaging

The Fourier inversion of phased coherent diffraction patterns offers images without the resolution and depth-of-focus limitations of lens-based tomographic systems. We report on our recent experimental images inverted using recent developments in phase retrieval algorithms, and summarize efforts that led to these accomplishments. These include ab-initio reconstruction of a two-dimensional test pattern, infinite depth of focus image of a thick object, and its high-resolution (~10 nm resolution) three-dimensional image. Developments on the structural imaging of low density aerogel samples are discussed.Comment: 5 pages, X-Ray Microscopy 2005, Himeji, Japa

Lipidic cubic phase serial millisecond crystallography using synchrotron radiation.

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins.Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven protonpump bacteriorhodopsin (bR) at a resolution of 2.4 A ° . The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway