Advantages of a synchrotron bending magnet as the sample illuminator for a wide-field X-ray microscope.

In this paper the choice between bending magnets and insertion devices as sample illuminators for a hard X-ray full-field microscope is investigated. An optimized bending-magnet beamline design is presented. Its imaging speed is very competitive with the performance of similar microscopes installed currently at insertion-device beamlines. The fact that imaging X-ray microscopes can accept a large phase space makes them very well suited to the output characteristics of bending magnets which are often a plentiful and paid-for resource. There exist opportunities at all synchrotron light sources to take advantage of this finding to build bending-magnet beamlines that are dedicated to transmission X-ray microscope facilities. It is expected that demand for such facilities will increase as three-dimensional tomography becomes routine and advanced techniques such as mosaic tomography and XANES tomography (taking three-dimensional tomograms at different energies to highlight elemental and chemical differences) become more widespread.

Pseudo-single-bunch with adjustable frequency: a new operation mode for synchrotron light sources.

We present the concept and results of pseudo-single-bunch (PSB) operation--a new operational mode at the advanced light source--that can greatly expand the capabilities of synchrotron light sources to carry out dynamics and time-of-flight experiments. In PSB operation, a single electron bunch is displaced transversely from the other electron bunches using a short-pulse, high-repetition-rate kicker magnet. Experiments that require light emitted only from a single bunch can stop the light emitted from the other bunches using a collimator. Other beam lines will only see a small reduction in flux due to the displaced bunch. As a result, PSB eliminates the need to schedule multibunch and timing experiments during different running periods. Furthermore, the time spacing of PSB pulses can be adjusted from milliseconds to microseconds with a novel "kick-and-cancel" scheme, which can significantly alleviate complications of using high-power choppers and substantially reduce the rate of sample damage.

An assessment of the resolution limitation due to radiation-damage in x-ray diffraction microscopy.

X-ray diffraction microscopy (XDM) is a new form of x-ray imaging that is being practiced at several third-generation synchrotron-radiation x-ray facilities. Nine years have elapsed since the technique was first introduced and it has made rapid progress in demonstrating high-resolution three-dimensional imaging and promises few-nm resolution with much larger samples than can be imaged in the transmission electron microscope. Both life- and materials-science applications of XDM are intended, and it is expected that the principal limitation to resolution will be radiation damage for life science and the coherent power of available x-ray sources for material science. In this paper we address the question of the role of radiation damage. We use a statistical analysis based on the so-called "dose fractionation theorem" of Hegerl and Hoppe to calculate the dose needed to make an image of a single life-science sample by XDM with a given resolution. We find that for simply-shaped objects the needed dose scales with the inverse fourth power of the resolution and present experimental evidence to support this finding. To determine the maximum tolerable dose we have assembled a number of data taken from the literature plus some measurements of our own which cover ranges of resolution that are not well covered otherwise. The conclusion of this study is that, based on the natural contrast between protein and water and "Rose-criterion" image quality, one should be able to image a frozen-hydrated biological sample using XDM at a resolution of about 10 nm.

The oversampling phasing method.

Sampling the diffraction pattern of a finite specimen more finely than the Nyquist frequency (the inverse of the size of the diffracting specimen) corresponds to surrounding the electron density of the specimen with a no-density region. When the no-density region is bigger than the electron-density region, sufficient information is recorded so that the phase information can be retrieved from the oversampled diffraction pattern, at least in principle. By employing an iterative algorithm, the phase information from the oversampled diffraction pattern of a micrometre-sized test specimen has been successfully retrieved. This method is believed to be able to open a door for high-resolution three-dimensional structure determination of complex and non-crystalline biological specimens, i.e. whole cells and sub-micrometre molecular clusters and micrometre-sized protein crystals. With the possible appearance in the future of X-ray free-electron lasers, it may become possible to image single molecules by recording diffraction patterns before radiation damage manifests itself.

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy.

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 microm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 1010 Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography (Wang et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc. 197, 80-93) or spectroscopic analysis.

Illumination for coherent soft X-ray applications: the new X1A beamline at the NSLS.

The X1A soft X-ray undulator beamline at the NSLS has been rebuilt to serve two microscopy stations operating simultaneously. Separate spherical-grating monochromators provide the resolving power required for XANES spectroscopy at the C, N and O absorption edges. The exit slits are fixed and define the coherent source for the experiments. The optical design and the operational performance are described.

Hemostatic methods for the management of spleen and liver injuries.

The spleen and liver are the most frequently injured organs during blunt and penetrating abdominal trauma. Emergency laparotomy is crucial for early control of bleeding and to prevent "secondary" injury as a result of physiologic splanchnic vasoconstriction and free oxygen radicals. Altogether 98 patients with spleen and liver injuries were treated over an 8-year period. Primary orthotopic spleen preservation could be achieved in 46 of 63 patients. In 58 patients with hepatic trauma, hemostatic treatment was chosen based on the severity of the injury. Nonoperative management was used for four splenic and seven hepatic trauma patients. The most commonly used techniques were fibrin sealing, suturing, and débridement for hepatic injury and mesh splenorrhaphy, fibrin glue, and partial resection with a TA stapler for splenic injury. The death of patients with complex injuries was mainly due to preclinical massive blood loss and multiple organ failure.

Mapping and measuring DNA to protein ratios in mammalian sperm head by XANES imaging.

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.

X-ray microscopy: preparations for studies of frozen hydrated specimens.

X-ray microscopes provide higher resolution than visible light microscopes. Wet, biological materials with a water thickness of up to about 10 microns can be imaged with good contrast using soft X-rays with wavelengths between the oxygen and carbon absorption edges (at 24 and 43 A). The Stony Brook group has developed and operates a scanning transmission X-ray microscope (STXM) at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The microscope is used for imaging with a current resolution of 50 nm, and for elemental and chemical state mapping. Radiation damage imposes a significant limitation upon high resolution X-ray microscopy of room temperature wet specimens. Experience from electron microscopy suggests that cryo techniques allow vitrified specimens to be imaged repeatedly. This is due to the increased radiation stability of biological specimens in the frozen hydrated state. Better radiation stability has been shown recently with a cryo transmission X-ray microscope developed by the University of Göttingen, operating at the BESSY storage ring in Berlin, Germany. At Stony Brook, we are developing a cryo scanning transmission X-ray microscope (CryoSTXM) to carry out imaging and spectro-microscopy experiments on frozen hydrated specimens. This article will give an outlook onto the research projects that we plan to perform using the CryoSTXM.

Chemical contrast in X-ray microscopy and spatially resolved XANES spectroscopy of organic specimens.

The scanning transmission x-ray microscope at the National Synchrotron Light Source has been used to record x-ray absorption near-edge structure (XANES) spectra from 0.01-square-micrometer regions of organic specimens. The spectral features observed reflect the molecular structure of the dominant absorbing atoms and provide the contrast mechanism for high-resolution imaging with chemical sensitivity. This technique was used with x-ray energies near the carbon K absorption edge to identify and map separate phases in various polymer blends and to map the DNA distribution in chromosomes with a spatial resolution of 55 nanometers.

High-Resolution Imaging by Fourier Transform X-ray Holography.

Fourier transform x-ray holography has been used to image gold test objects with submicrometer structure, resolving features as small as 60 nanometers. The hologram-recording instrument uses coherent 3.4-nanometer radiation from the soft x-ray undulator beamline X1A at the National Synchrotron Light Source. The specimen to be imaged is placed near the first-order focal spot produced by a Fresnel zone plate; the other orders, chiefly the zeroth, illuminate the specimen. The wave scattered by the specimen interferes with the spherical reference wave from the focal spot, forming a hologram with fringes of low spatial frequency. The hologram is recorded in digital form by a charge-coupled device camera, and the specimen image is obtained by numerical reconstruction.

Coherent Radiation for X-Ray Imaging-The Soft X-Ray Undulator and the X1A Beamline at the NSLS.

An undulator-based beamline was built and commissioned at the National Synchrotron Light Source to provide tunable coherent radiation in the 200-800 eV range. The low emittance of the storage ring means that the undulator source has high brightness so that a large flux of coherent x rays is delivered to experimental stations. The beamline uses a horizontally dispersing bichromator that allows two experiments to run simultaneously, making use of the first and second harmonics of the undulator output. In addition, the use of horizontally deflecting optics enables the beamline alignment to be insensitive to electron beam motion since the horizontal electron beam size is quite large. The beamline and its performance are discussed with emphasis on the optics and on stability, radiation, and vacuum considerations.

The interior of a whole and unmodified biological object--the zymogen granule--viewed with a high-resolution X-ray microscope.

We report the ability of focused soft X-rays to visualize at spatial resolution well beyond that of the optical microscope (less than 100 nm) the interior of a small, whole biological object without fixation, staining, dehydration or sectioning. Quantitative estimation of its protein content with unique femtogram sensitivity is also reported. The present results represent a significant step towards the goals of natural imaging and chemical mapping of biological structures with soft X-rays.