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1.  X-ray near-field speckle: implementation and critical analysis 
Journal of Synchrotron Radiation  2011;18(Pt 6):823-834.
A coherence-based X-ray near-field speckle detector has been implemented and characterized for its capability of studying static structure and dynamics.
The newly introduced coherence-based technique of X-ray near-field speckle (XNFS) has been implemented at 8-ID-I at the Advanced Photon Source. In the near-field regime of high-brilliance synchrotron X-rays scattered from a sample of interest, it turns out that, when the scattered radiation and the main beam both impinge upon an X-ray area detector, the measured intensity shows low-contrast speckles, resulting from interference between the incident and scattered beams. A micrometer-resolution XNFS detector with a high numerical aperture microscope objective has been built and its capability for studying static structures and dynamics at longer length scales than traditional far-field X-ray scattering techniques is demonstrated. Specifically, the dynamics of dilute silica and polystyrene colloidal samples are characterized. This study reveals certain limitations of the XNFS technique, especially in the characterization of static structures, which is discussed.
PMCID: PMC3258091  PMID: 21997906
X-ray; near field; speckle; spectroscopy; scattering
2.  Oversampling smoothness: an effective algorithm for phase retrieval of noisy diffraction intensities1  
Journal of Applied Crystallography  2013;46(Pt 2):312-318.
An iterative phase retrieval algorithm, termed oversampling smoothness (OSS), has been developed to reconstruct fine features in weakly scattered objects such as biological specimens from noisy experimental data. OSS is expected to find application in the rapidly growing coherent diffraction imaging field as well as other disciplines where phase retrieval from noisy Fourier magnitudes is needed.
Coherent diffraction imaging (CDI) is high-resolution lensless microscopy that has been applied to image a wide range of specimens using synchrotron radiation, X-ray free-electron lasers, high harmonic generation, soft X-ray lasers and electrons. Despite recent rapid advances, it remains a challenge to reconstruct fine features in weakly scattering objects such as biological specimens from noisy data. Here an effective iterative algorithm, termed oversampling smoothness (OSS), for phase retrieval of noisy diffraction intensities is presented. OSS exploits the correlation information among the pixels or voxels in the region outside of a support in real space. By properly applying spatial frequency filters to the pixels or voxels outside the support at different stages of the iterative process (i.e. a smoothness constraint), OSS finds a balance between the hybrid input–output (HIO) and error reduction (ER) algorithms to search for a global minimum in solution space, while reducing the oscillations in the reconstruction. Both numerical simulations with Poisson noise and experimental data from a biological cell indicate that OSS consistently outperforms the HIO, ER–HIO and noise robust (NR)–HIO algorithms at all noise levels in terms of accuracy and consistency of the reconstructions. It is expected that OSS will find application in the rapidly growing CDI field, as well as other disciplines where phase retrieval from noisy Fourier magnitudes is needed. The MATLAB (The MathWorks Inc., Natick, MA, USA) source code of the OSS algorithm is freely available from
PMCID: PMC3627409  PMID: 23596339
coherent diffraction imaging; lensless imaging; oversampling; phase retrieval; image reconstruction; X-ray free-electron lasers
3.  Real-time and Sub-wavelength Ultrafast Coherent Diffraction Imaging in the Extreme Ultraviolet 
Scientific Reports  2014;4:7356.
Coherent Diffraction Imaging is a technique to study matter with nanometer-scale spatial resolution based on coherent illumination of the sample with hard X-ray, soft X-ray or extreme ultraviolet light delivered from synchrotrons or more recently X-ray Free-Electron Lasers. This robust technique simultaneously allows quantitative amplitude and phase contrast imaging. Laser-driven high harmonic generation XUV-sources allow table-top realizations. However, the low conversion efficiency of lab-based sources imposes either a large scale laser system or long exposure times, preventing many applications. Here we present a lensless imaging experiment combining a high numerical aperture (NA = 0.8) setup with a high average power fibre laser driven high harmonic source. The high flux and narrow-band harmonic line at 33.2 nm enables either sub-wavelength spatial resolution close to the Abbe limit (Δr = 0.8λ) for long exposure time, or sub-70 nm imaging in less than one second. The unprecedented high spatial resolution, compactness of the setup together with the real-time capability paves the way for a plethora of applications in fundamental and life sciences.
PMCID: PMC4258652  PMID: 25483626
4.  Radiation-induced melting in coherent X-ray diffractive imaging at the nanoscale 
Journal of Synchrotron Radiation  2011;18(Pt 4):580-594.
Coherent X-ray diffraction techniques play an increasingly significant role in imaging nanoscale structures which range from metallic and semiconductor samples to biological objects. The conventional knowledge about radiation damage effects caused by ever higher brilliance X-ray sources has to be critically revised while studying nanostructured materials.
Coherent X-ray diffraction techniques play an increasingly significant role in the imaging of nanoscale structures, ranging from metallic and semiconductor to biological objects. In material science, X-rays are usually considered to be of a low-destructive nature, but under certain conditions they can cause significant radiation damage and heat loading on the samples. The qualitative literature data concerning the tolerance of nanostructured samples to synchrotron radiation in coherent diffraction imaging experiments are scarce. In this work the experimental evidence of a complete destruction of polymer and gold nanosamples by the synchrotron beam is reported in the case of imaging at 1–10 nm spatial resolution. Numerical simulations based on a heat-transfer model demonstrate the high sensitivity of temperature distribution in samples to macroscopic experimental parameters such as the conduction properties of materials, radiation heat transfer and convection. However, for realistic experimental conditions the calculated rates of temperature rise alone cannot explain the melting transitions observed in the nanosamples. Comparison of these results with the literature data allows a specific scenario of the sample destruction in each particular case to be presented, and a strategy for damage reduction to be proposed.
PMCID: PMC3286865  PMID: 21685675
coherent X-ray diffraction imaging; high-resolution synchrotron radiation; heat load; nanosize effects
5.  Determination of the effect of source intensity profile on speckle contrast using coherent spatial frequency domain imaging 
Biomedical Optics Express  2012;3(6):1340-1349.
Laser Speckle Imaging (LSI) is fast, noninvasive technique to image particle dynamics in scattering media such as biological tissue. While LSI measurements are independent of the overall intensity of the laser source, we find that spatial variations in the laser source profile can impact measured flow rates. This occurs due to differences in average photon path length across the profile, and is of significant concern because all lasers have some degree of natural Gaussian profile in addition to artifacts potentially caused by projecting optics. Two in vivo measurement are performed to show that flow rates differ based on location with respect to the beam profile. A quantitative analysis is then done through a speckle contrast forward model generated within a coherent Spatial Frequency Domain Imaging (cSFDI) formalism. The model predicts remitted speckle contrast as a function of spatial frequency, optical properties, and scattering dynamics. Comparison with experimental speckle contrast images were done using liquid phantoms with known optical properties for three common beam shapes. cSFDI is found to accurately predict speckle contrast for all beam shapes to within 5% root mean square error. Suggestions for improving beam homogeneity are given, including a widening of the natural beam Gaussian, proper diffusing glass spreading, and flat top shaping using microlens arrays.
PMCID: PMC3370974  PMID: 22741080
(110.6150) Speckle imaging; (170.3660) Light propagation in tissues
6.  Spatial Harmonic Imaging of X-ray Scattering—Initial Results 
Coherent X-ray scattering is related to the electron density distribution by a Fourier transform, and therefore a window into the microscopic structures of biological samples. Current techniques of scattering rely on small-angle measurements from highly collimated X-ray beams produced from synchrotron light sources. Imaging of the distribution of scattering provides a new contrast mechanism which is different from absorption radiography, but is a lengthy process of raster or flue scans of the beam over the object. Here, we describe an imaging technique in the spatial frequency domain capable of acquiring both the scattering and absorption distributions in a single exposure. We present first results obtained with conventional X-ray equipment. This method interposes a grid between the X-ray source and the imaged object, so that the grid-modulated image contains a primary image and a grid harmonic image. The ratio between the harmonic and primary images is shown to be a pure scattering image. It is the auto-correlation of the electron density distribution at a specific distance. We tested a number of samples at 60–200 nm autocorrelation distance, and found the scattering images to be distinct from the absorption images and reveal new features. This technique is simple to implement, and should help broaden the imaging applications of X-ray scattering.
PMCID: PMC2882966  PMID: 18672418
Diffraction; imaging; scatter; X-ray
7.  In situ micro-focused X-ray beam characterization with a lensless camera using a hybrid pixel detector 
Journal of Synchrotron Radiation  2014;21(Pt 2):333-339.
Position and size measurements of a micro-focused X-ray beam, using an X-ray beam imaging device based on a lensless camera that collects radiation scattered from a thin foil placed in the path of the beam at an oblique angle, are reported.
Results of studies on micro-focused X-ray beam diagnostics using an X-ray beam imaging (XBI) instrument based on the idea of recording radiation scattered from a thin foil of a low-Z material with a lensless camera are reported. The XBI instrument captures magnified images of the scattering region within the foil as illuminated by the incident beam. These images contain information about beam size, beam position and beam intensity that is extracted during dedicated signal processing steps. In this work the use of the device with beams for which the beam size is significantly smaller than that of a single detector pixel is explored. The performance of the XBI device equipped with a state-of-the-art hybrid pixel X-ray imaging sensor is analysed. Compared with traditional methods such as slit edge or wire scanners, the XBI micro-focused beam characterization is significantly faster and does not interfere with on-going experiments. The challenges associated with measuring micrometre-sized beams are described and ways of optimizing the resolution of beam position and size measurements of the XBI instrument are discussed.
PMCID: PMC3945420  PMID: 24562554
X-ray imaging; pinhole camera; beam diagnostics; micro-focus; scattering measurements; beam size measurements
8.  Processing of projections containing phase contrast in laboratory micro-computerized tomography imaging 
Journal of Applied Crystallography  2013;46(Pt 4):933-938.
Processing of phase-contrast images in laboratory conditions is described.
Free-space-propagation-based imaging belongs to several techniques for achieving phase contrast in the hard X-ray range. The basic precondition is to use an X-ray beam with a high degree of coherence. Although the best sources of coherent X-rays are synchrotrons, spatially coherent X-rays emitted from a sufficiently small spot of laboratory microfocus or sub-microfocus sources allow the transfer of some of the modern imaging techniques from synchrotrons to laboratories. Spatially coherent X-rays traverse a sample leading to a phase shift. Beam deflection induced by the local change of refractive index may be expressed as a dark–bright contrast on the edges of the object in an X-ray projection. This phenomenon of edge enhancement leads to an increase in spatial resolution of X-ray projections but may also lead to unpleasant artefacts in computerized tomography unless phase and absorption contributions are separated. The possibilities of processing X-ray images of lightweight objects containing phase contrast using phase-retrieval methods in laboratory conditions are tested and the results obtained are presented. For this purpose, simulated and recorded X-ray projections taken from a laboratory imaging system with a microfocus X-ray source and a high-resolution CCD camera were processed and a qualitative comparison of results was made.
PMCID: PMC3769065  PMID: 24046501
phase-contrast imaging; X-ray imaging; X-ray radiography; digital radiography; computerized tomography; computed radiography
9.  Demonstration of Feasibility of X-Ray Free Electron Laser Studies of Dynamics of Nanoparticles in Entangled Polymer Melts 
Scientific Reports  2014;4:6017.
The recent advent of hard x-ray free electron lasers (XFELs) opens new areas of science due to their exceptional brightness, coherence, and time structure. In principle, such sources enable studies of dynamics of condensed matter systems over times ranging from femtoseconds to seconds. However, the studies of “slow” dynamics in polymeric materials still remain in question due to the characteristics of the XFEL beam and concerns about sample damage. Here we demonstrate the feasibility of measuring the relaxation dynamics of gold nanoparticles suspended in polymer melts using X-ray photon correlation spectroscopy (XPCS), while also monitoring eventual X-ray induced damage. In spite of inherently large pulse-to-pulse intensity and position variations of the XFEL beam, measurements can be realized at slow time scales. The X-ray induced damage and heating are less than initially expected for soft matter materials.
PMCID: PMC4127496  PMID: 25109363
10.  Multimodal hard X-ray imaging of a mammography phantom at a compact synchrotron light source 
Journal of Synchrotron Radiation  2012;19(Pt 4):525-529.
Dose-compatible measurements of a mammography phantom demonstrate an increase in contrast attainable through differential phase and dark-field imaging over conventional absorption-based projections. All measurements have been performed at 21 keV using a monochromatic laser-driven miniature synchrotron X-ray source (Compact Light Source).
The Compact Light Source is a miniature synchrotron producing X-rays at the interaction point of a counter-propagating laser pulse and electron bunch through the process of inverse Compton scattering. The small transverse size of the luminous region yields a highly coherent beam with an angular divergence of a few milliradians. The intrinsic monochromaticity and coherence of the produced X-rays can be exploited in high-sensitivity differential phase-contrast imaging with a grating-based interferometer. Here, the first multimodal X-ray imaging experiments at the Compact Light Source at a clinically compatible X-ray energy of 21 keV are reported. Dose-compatible measurements of a mammography phantom clearly demonstrate an increase in contrast attainable through differential phase and dark-field imaging over conventional attenuation-based projections.
PMCID: PMC3380656  PMID: 22713884
medical X-ray imaging; phase contrast; inverse Compton X-rays
11.  Single mimivirus particles intercepted and imaged with an X-ray laser 
Seibert, M. Marvin | Ekeberg, Tomas | Maia, Filipe R. N. C. | Svenda, Martin | Andreasson, Jakob | Jönsson, Olof | Odić, Duško | Iwan, Bianca | Rocker, Andrea | Westphal, Daniel | Hantke, Max | DePonte, Daniel P. | Barty, Anton | Schulz, Joachim | Gumprecht, Lars | Coppola, Nicola | Aquila, Andrew | Liang, Mengning | White, Thomas A. | Martin, Andrew | Caleman, Carl | Stern, Stephan | Abergel, Chantal | Seltzer, Virginie | Claverie, Jean-Michel | Bostedt, Christoph | Bozek, John D. | Boutet, Sébastien | Miahnahri, A. Alan | Messerschmidt, Marc | Krzywinski, Jacek | Williams, Garth | Hodgson, Keith O. | Bogan, Michael J. | Hampton, Christina Y. | Sierra, Raymond G. | Starodub, Dmitri | Andersson, Inger | Bajt, Saša | Barthelmess, Miriam | Spence, John C. H. | Fromme, Petra | Weierstall, Uwe | Kirian, Richard | Hunter, Mark | Doak, R. Bruce | Marchesini, Stefano | Hau-Riege, Stefan P. | Frank, Matthias | Shoeman, Robert L. | Lomb, Lukas | Epp, Sascha W. | Hartmann, Robert | Rolles, Daniel | Rudenko, Artem | Schmidt, Carlo | Foucar, Lutz | Kimmel, Nils | Holl, Peter | Rudek, Benedikt | Erk, Benjamin | Hömke, André | Reich, Christian | Pietschner, Daniel | Weidenspointner, Georg | Strüder, Lothar | Hauser, Günter | Gorke, Hubert | Ullrich, Joachim | Schlichting, Ilme | Herrmann, Sven | Schaller, Gerhard | Schopper, Florian | Soltau, Heike | Kühnel, Kai-Uwe | Andritschke, Robert | Schröter, Claus-Dieter | Krasniqi, Faton | Bott, Mario | Schorb, Sebastian | Rupp, Daniela | Adolph, Marcus | Gorkhover, Tais | Hirsemann, Helmut | Potdevin, Guillaume | Graafsma, Heinz | Nilsson, Björn | Chapman, Henry N. | Hajdu, Janos
Nature  2011;470(7332):78-81.
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1–4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
PMCID: PMC4038304  PMID: 21293374
12.  Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function 
BMC Biology  2007;5:6.
Despite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.
We evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 μW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion.
Synchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.
PMCID: PMC1831761  PMID: 17331247
13.  Small-angle scattering computed tomography (SAS-CT) using a Talbot-Lau interferometer and a rotating anode x-ray tube: theory and experiments 
Optics express  2010;18(12):12960-12970.
X-ray differential phase contrast imaging methods, including projection imaging and the corresponding computed tomography (CT), have been implemented using a Talbot interferometer and either a synchrotron beam line or a low brilliance x-ray source generated by a stationary-anode x-ray tube. From small-angle scattering events which occur as an x-ray propagates through a medium, a signal intensity loss can be recorded and analyzed for an understanding of the micro-structures in an image object. This has been demonstrated using a Talbot-Lau interferometer and a stationary-anode x-ray tube. In this paper, theoretical principles and an experimental implementation of the corresponding CT imaging method are presented. First, a line integral is derived from analyzing the cross section of the small-angle scattering events. This method is referred to as small-angle scattering computed tomography (SAS-CT). Next, a Talbot-Lau interferometer and a rotating-anode x-ray tube were used to implement SAS-CT. A physical phantom and human breast tissue sample were used to demonstrate the reconstructed SAS-CT image volumes.
PMCID: PMC3746741  PMID: 20588425
14.  Mini-beam collimator enables microcrystallography experiments on standard beamlines 
Journal of Synchrotron Radiation  2009;16(Pt 2):217-225.
A ‘mini-beam’ apparatus has been developed that conditions the size of an X-ray beam to 5 µm. The design of the apparatus and the characterization of the focal size and flux are presented.
The high-brilliance X-ray beams from undulator sources at third-generation synchrotron facilities are excellent tools for solving crystal structures of important and challenging biological macromolecules and complexes. However, many of the most important structural targets yield crystals that are too small or too inhomogeneous for a ‘standard’ beam from an undulator source, ∼25–50 µm (FWHM) in the vertical and 50–100 µm in the horizontal direction. Although many synchrotron facilities have microfocus beamlines for other applications, this capability for macromolecular crystallography was pioneered at ID-13 of the ESRF. The National Institute of General Medical Sciences and National Cancer Institute Collaborative Access Team (GM/CA-CAT) dual canted undulator beamlines at the APS deliver high-intensity focused beams with a minimum focal size of 20 µm × 65 µm at the sample position. To meet growing user demand for beams to study samples of 10 µm or less, a ‘mini-beam’ apparatus was developed that conditions the focused beam to either 5 µm or 10 µm (FWHM) diameter with high intensity. The mini-beam has a symmetric Gaussian shape in both the horizontal and vertical directions, and reduces the vertical divergence of the focused beam by 25%. Significant reduction in background was achieved by implementation of both forward- and back-scatter guards. A unique triple-collimator apparatus, which has been in routine use on both undulator beamlines since February 2008, allows users to rapidly interchange the focused beam and conditioned mini-beams of two sizes with a single mouse click. The device and the beam are stable over many hours of routine operation. The rapid-exchange capability has greatly facilitated sample screening and resulted in several structures that could not have been obtained with the larger focused beam.
PMCID: PMC2725011  PMID: 19240333
mini-beam; microbeam; microdiffraction; macromolecular crystallography
15.  Time-resolved coherent X-ray diffraction imaging of surface acoustic waves 
Journal of Applied Crystallography  2014;47(Pt 5):1596-1605.
The time-dependent one-dimensional height profile of a standing surface acoustic wave on an LiNbO3 substrate has been reconstructed from stroboscopically recorded coherent grazing-incidence small-angle diffraction patterns.
Time-resolved coherent X-ray diffraction experiments of standing surface acoustic waves, illuminated under grazing incidence by a nanofocused synchrotron beam, are reported. The data have been recorded in stroboscopic mode at controlled and varied phase between the acoustic frequency generator and the synchrotron bunch train. At each time delay (phase angle), the coherent far-field diffraction pattern in the small-angle regime is inverted by an iterative algorithm to yield the local instantaneous surface height profile along the optical axis. The results show that periodic nanoscale dynamics can be imaged at high temporal resolution in the range of 50 ps (pulse length).
PMCID: PMC4180738  PMID: 25294979
coherent X-ray diffraction imaging; surface acoustic waves; nanostructure
16.  Coherent diffraction microscopy at SPring-8: instrumentation, data acquisition and data analysis 
Journal of Synchrotron Radiation  2011;18(Pt 2):293-298.
An instrumentation and data analysis review of coherent diffraction microscopy at SPring-8 is given. This work will be of interest to those who want to apply coherent diffraction imaging to studies of materials science and biological samples.
Since the first demonstration of coherent diffraction microscopy in 1999, this lensless imaging technique has been experimentally refined by continued developments. Here, instrumentation and experimental procedures for measuring oversampled diffraction patterns from non-crystalline specimens using an undulator beamline (BL29XUL) at SPring-8 are presented. In addition, detailed post-experimental data analysis is provided that yields high-quality image reconstructions. As the acquisition of high-quality diffraction patterns is at least as important as the phase-retrieval procedure to guarantee successful image reconstructions, this work will be of interest for those who want to apply this imaging technique to materials science and biological samples.
PMCID: PMC3042331  PMID: 21335919
coherent diffraction microscopy; coherent diffraction imaging; lensless imaging; oversampling; phase retrieval
17.  X-Ray Imaging of Poly(Ethylene Glycol) Hydrogels Without Contrast Agents 
Tissue Engineering. Part C, Methods  2010;16(6):1597-1600.
Hydrogels have shown promise for a number of tissue engineering applications. However, their high water content results in little or no image contrast when using conventional X-ray imaging techniques. X-ray imaging techniques based on phase-contrast have shown promise for biomedical application due to their ability to provide information about the X-ray refraction properties of samples. Nonporous and porous poly(ethylene glycol) hydrogels were synthesized and imaged using a synchrotron light source employing a silicon analyzer crystal and an X-ray energy of 40-keV. Data were acquired at 21 angular analyzer positions spanning the range of −5 to 5 μrad. Images that depict the projected X-ray absorption, refraction, and ultra-small-angle scatter (USAXS) properties of the hydrogels were reconstructed from the measurement data. The poly(ethylene glycol) hydrogels could be discerned from surrounding water and soft tissue in the refraction image but not the absorption or USAXS images. In addition, the refraction images of the porous hydrogels have a speckle pattern resulting in increased image texture in comparison to nonporous hydrogels. To our knowledge, this is the first study to show that X-ray phase-contrast imaging techniques can identify and provide detail on hydrogel structure without the addition of contrast agents.
PMCID: PMC2988626  PMID: 20662738
18.  Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells 
Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques.
PMCID: PMC2839371  PMID: 19324670
cartilage; chondrocyte; synchrotron micro-computed tomography; histology; scanning electron microscopy; three-dimensional imaging
19.  Evaluation and Correction for Optical Scattering Variations in Laser Speckle Rheology of Biological Fluids 
PLoS ONE  2013;8(5):e65014.
Biological fluids fulfill key functionalities such as hydrating, protecting, and nourishing cells and tissues in various organ systems. They are capable of these versatile tasks owing to their distinct structural and viscoelastic properties. Characterizing the viscoelastic properties of bio-fluids is of pivotal importance for monitoring the development of certain pathologies as well as engineering synthetic replacements. Laser Speckle Rheology (LSR) is a novel optical technology that enables mechanical evaluation of tissue. In LSR, a coherent laser beam illuminates the tissue and temporal speckle intensity fluctuations are analyzed to evaluate mechanical properties. The rate of temporal speckle fluctuations is, however, influenced by both optical and mechanical properties of tissue. Therefore, in this paper, we develop and validate an approach to estimate and compensate for the contributions of light scattering to speckle dynamics and demonstrate the capability of LSR for the accurate extraction of viscoelastic moduli in phantom samples and biological fluids of varying optical and mechanical properties.
PMCID: PMC3660338  PMID: 23705028
20.  X-ray pump optical probe cross-correlation study of GaAs 
Nature photonics  2012;6(2):111-114.
Ultrafast dynamics in atomic, molecular and condensed-matter systems are increasingly being studied using optical-pump, X-ray probe techniques where subpicosecond laser pulses excite the system and X-rays detect changes in absorption spectra and local atomic structure1–3. New opportunities are appearing as a result of improved synchrotron capabilities and the advent of X-ray free-electron lasers4,5. These source improvements also allow for the reverse measurement: X-ray pump followed by optical probe. We describe here how an X-ray pump beam transforms a thin GaAs specimen from a strong absorber into a nearly transparent window in less than 100 ps, for laser photon energies just above the bandgap. We find the opposite effect—X-ray induced optical opacity—for photon energies just below the bandgap. This raises interesting questions about the ultrafast many-body response of semiconductors to X-ray absorption, and provides a new approach for an X-ray/optical cross-correlator for synchrotron and X-ray free-electron laser applications.
PMCID: PMC3418921  PMID: 22899965
21.  High-dynamic-range coherent diffractive imaging: ptychography using the mixed-mode pixel array detector 
Journal of Synchrotron Radiation  2014;21(Pt 5):1167-1174.
The advantages of a novel wide dynamic range hard X-ray detector are demonstrated for (ptychographic) coherent X-ray diffractive imaging.
Coherent (X-ray) diffractive imaging (CDI) is an increasingly popular form of X-ray microscopy, mainly due to its potential to produce high-resolution images and the lack of an objective lens between the sample and its corresponding imaging detector. One challenge, however, is that very high dynamic range diffraction data must be collected to produce both quantitative and high-resolution images. In this work, hard X-ray ptychographic coherent diffractive imaging has been performed at the P10 beamline of the PETRA III synchrotron to demonstrate the potential of a very wide dynamic range imaging X-ray detector (the Mixed-Mode Pixel Array Detector, or MM-PAD). The detector is capable of single photon detection, detecting fluxes exceeding 1 × 108 8-keV photons pixel−1 s−1, and framing at 1 kHz. A ptychographic reconstruction was performed using a peak focal intensity on the order of 1 × 1010 photons µm−2 s−1 within an area of approximately 325 nm × 603 nm. This was done without need of a beam stop and with a very modest attenuation, while ‘still’ images of the empty beam far-field intensity were recorded without any attenuation. The treatment of the detector frames and CDI methodology for reconstruction of non-sensitive detector regions, partially also extending the active detector area, are described.
PMCID: PMC4151683  PMID: 25178008
pixel array detectors; coherent X-ray diffractive imaging; ptychography
22.  Fourier phase microscopy with white light 
Biomedical Optics Express  2013;4(8):1434-1441.
Laser-based Fourier phase microscopy (FPM) works on the principle of decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other. However, due to the coherent illumination, the contrast in phase images is degraded by speckles. In this paper we present FPM with spatially coherent white light (wFPM), which offers high spatial phase sensitivity due to the low temporal coherence and high temporal phase stability due to common path geometry. Further, by using a fast spatial light modulator (SLM) and a fast scientific-grade complementary metal oxide semiconductor (sCMOS) camera, we report imaging at a maximum rate of 12.5 quantitative phase frames per second with 5.5 mega pixels image size. We illustrate the utility of wFPM as a contrast enhancement as well as dynamic phase measurement method by imaging section of benign colonic glands and red blood cell membrane fluctuation.
PMCID: PMC3756570  PMID: 24010005
(170.0180) Microscopy; (070.0070) Fourier optics and signal processing; (070.6120) Spatial light modulators; (120.5050) Phase measurement
23.  Incorrect support and missing center tolerances of phasing algorithms 
Optics express  2010;18(25):26441-26449.
In x-ray diffraction microscopy, iterative algorithms retrieve reciprocal space phase information, and a real space image, from an object's coherent diffraction intensities through the use of a priori information such as a finite support constraint. In many experiments, the object's shape or support is not well known, and the diffraction pattern is incompletely measured. We describe here computer simulations to look at the effects of both of these possible errors when using several common reconstruction algorithms. Overly tight object supports prevent successful convergence; however, we show that this can often be recognized through pathological behavior of the phase retrieval transfer function. Dynamic range limitations often make it difficult to record the central speckles of the diffraction pattern. We show that this leads to increasing artifacts in the image when the number of missing central speckles exceeds about 10, and that the removal of unconstrained modes from the reconstructed image is helpful only when the number of missing central speckles is less than about 50. This simulation study helps in judging the reconstructability of experimentally recorded coherent diffraction patterns.
PMCID: PMC3068748  PMID: 21164994
24.  Three-dimensional motion correction using speckle and phase for in vivo computed optical interferometric tomography 
Biomedical Optics Express  2014;5(12):4131-4143.
Over the years, many computed optical interferometric techniques have been developed to perform high-resolution volumetric tomography. By utilizing the phase and amplitude information provided with interferometric detection, post-acquisition corrections for defocus and optical aberrations can be performed. The introduction of the phase, though, can dramatically increase the sensitivity to motion (most prominently along the optical axis). In this paper, we present two algorithms which, together, can correct for motion in all three dimensions with enough accuracy for defocus and aberration correction in computed optical interferometric tomography. The first algorithm utilizes phase differences within the acquired data to correct for motion along the optical axis. The second algorithm utilizes the addition of a speckle tracking system using temporally- and spatially-coherent illumination to measure motion orthogonal to the optical axis. The use of coherent illumination allows for high-contrast speckle patterns even when imaging apparently uniform samples or when highly aberrated beams cannot be avoided.
PMCID: PMC4285593  PMID: 25574426
(100.5090) Phase-only filters; (110.3010) Image reconstruction techniques; (110.3175) Interferometric imaging; (110.3200) Inverse scattering; (110.4280) Noise in imaging systems; (110.4500) Optical coherence tomography
25.  GENFIT: software for the analysis of small-angle X-ray and neutron scattering data of macro­molecules in solution 
Journal of Applied Crystallography  2014;47(Pt 3):1132-1139.
GENFIT is a new computer code featuring an advanced model-fitting capability to analyse small-angle X-ray and neutron scattering data of macromolecular systems. Batches of experimental curves can be simultaneously best fitted using common parameters issued from combinations of models and, if applicable, constrained by physical and/or phenomenological relations.
Many research topics in the fields of condensed matter and the life sciences are based on small-angle X-ray and neutron scattering techniques. With the current rapid progress in source brilliance and detector technology, high data fluxes of ever-increasing quality are produced. In order to exploit such a huge quantity of data and richness of information, wider and more sophisticated approaches to data analysis are needed. Presented here is GENFIT, a new software tool able to fit small-angle scattering data of randomly oriented macromolecular or nanosized systems according to a wide list of models, including form and structure factors. Batches of curves can be analysed simultaneously in terms of common fitting parameters or by expressing the model parameters via physical or phenomenological link functions. The models can also be combined, enabling the user to describe complex heterogeneous systems.
PMCID: PMC4038801  PMID: 24904247
GENFIT; small-angle X-ray scattering; small-angle neutron scattering; macromolecules

Results 1-25 (843214)