X-ray fluorescence tomography involves the acquisition of a series of 2D x-ray fluorescence datasets between which a specimen is rotated. At the Advanced Photon Source at Argonne National Laboratory, the workflow at beamlines 2-ID-E and 21-ID-D (the Bionanoprobe, a cryogenic microscope system) has included the use of the program MAPS for obtaining elemental concentrations from 2D images, and the program TomoPy which was developed to include several tomographic reconstruction methods for x-ray transmission data. In the past, fluorescence projection images from an individual chemical element were hand-assembled into a 3D dataset for reconstruction using interactive tools such as ImageJ. We describe here the program MAPSToTomoPy, which provides a graphical user interface (GUI) to control a workflow between MAPS and TomoPy, with tools for visualizing the sinograms of projection image sequences from particular elements and to use these to help correct misalignments of the rotation axis. The program also provides an integrated output of the 3D distribution of the detected elements for subsequent 3D visualization packages.
X-ray fluorescence offers unparalleled sensitivity for imaging the nanoscale distribution of trace elements in micrometer thick samples, while x-ray ptychography offers an approach to image weakly fluorescing lighter elements at a resolution beyond that of the x-ray lens used. These methods can be used in combination, and in continuous scan mode for rapid data acquisition when using multiple probe mode reconstruction methods. We discuss here the opportunities and limitations of making use of additional information provided by ptychography to improve x-ray fluorescence images in two ways: by using position-error-correction algorithms to correct for scan distortions in fluorescence scans, and by considering the signal-to-noise limits on previously-demonstrated ptychographic probe deconvolution methods. This highlights the advantages of using a combined approach.
Hard X-ray fluorescence (XRF) microscopy offers unparalleled sensitivity for quantitative analysis of most of the trace elements in biological samples, such as Fe, Cu, and Zn. These trace elements play critical roles in many biological processes. With the advanced nano-focusing optics, nowadays hard X-rays can be focused down to 30 nm or below and can probe trace elements within subcellular compartments. However, XRF imaging does not usually reveal much information on ultrastructure, because the main constituents of biomaterials, i.e. H, C, N, and O, have low fluorescence yield and little absorption contrast at multi-keV X-ray energies. An alternative technique for imaging ultrastructure is ptychography. One can record far-field diffraction patterns from a coherently illuminated sample, and then reconstruct the complex transmission function of the sample. In theory the spatial resolution of ptychography can reach the wavelength limit. In this manuscript, we will describe the implementation of ptychography at the Bionanoprobe (a recently developed hard XRF nanoprobe at the Advanced Photon Source) and demonstrate simultaneous ptychographic and XRF imaging of frozen-hydrated biological whole cells. This method allows locating trace elements within the subcellular structures of biological samples with high spatial resolution. Additionally, both ptychographic and XRF imaging are compatible with tomographic approach for 3D visualization.
Analysis of large tomographic datasets at synchrotron light sources is becoming progressively more challenging due to the increasing data acquisition rates that new technologies in X-ray sources and detectors enable. The next generation of synchrotron facilities that are currently under design or construction throughout the world will provide diffraction limited X-ray sources and is expected to boost the current data rates by several orders of magnitude and stressing the need for the development and integration of efficient analysis tools more than ever. Here we describe in detail an attempt to provide such a collaborative framework for the analysis of synchrotron tomographic data that has the potential to unify the effort of different facilities and beamlines performing similar tasks. The proposed Python/C++ based framework is open-source, OS and data format independent, parallelizable and supports functional programming that many researchers prefer. This collaborative platform will affect all major synchrotron facilities where new effort is now dedicated into developing new tools that can be deployed at the facility for real time processing as well as distributed to users for off site data processing.
The Advanced Photon Source is currently developing a suite of new hard x-ray beamlines, aimed primarily at the study
of materials and devices under real conditions. One of the flagship beamlines of the APS Upgrade is the In-Situ
Nanoprobe beamline (ISN beamline), which will provide in-situ and operando characterization of advanced energy
materials and devices under change of temperature and gases, under applied fields, in 3D.
The ISN beamline is designed to deliver spatially coherent x-rays with photon energies between 4 keV and 30 keV to the
ISN instrument. As an x-ray source, a revolver-type undulator with two interchangeable magnetic structures,
optimized to provide high brilliance throughout the range of photon energies of 4 keV – 30 keV, will be used. The
ISN instrument will provide a smallest hard x-ray spot of 20 nm using diffractive optics, with sensitivity to sub-10
nm sample structures using coherent diffraction. Using nanofocusing mirrors in Kirkpatrick-Baez geometry, the ISN
will also provide a focus of 50 nm with a flux of 8·1011 Photons/s at a photon energy of 10 keV, several orders of
magnitude larger than what is currently available. This will allow imaging of trace amounts of most elements in the
periodic table, with a sensitivity to well below 100 atoms for most metals in thin samples. It will also enable nanospectroscopic
studies of the chemical state of most materials relevant to energy science. The ISN beamline will be
primarily used to study inorganic and organic photovoltaic systems, advanced batteries and fuel cells, nanoelectronics devices, and materials and systems diesigned to reduce the environmental impact of combustion.
Hard X-ray fluorescence microscopy is one of the most sensitive techniques to perform trace elemental analysis of
unsectioned biological samples, such as cells and tissues. As the spatial resolution increases beyond sub-micron
scale, conventional sample preparation method, which involves dehydration, may not be sufficient for preserving
subcellular structures in the context of radiation-induced artifacts. Imaging of frozen-hydrated samples under
cryogenic conditions is the only reliable way to fully preserve the three dimensional structures of the samples while
minimizing the loss of diffusible ions. To allow imaging under this hydrated “natural-state” condition, we have
developed the Bionanoprobe (BNP), a hard X-ray fluorescence nanoprobe with cryogenic capabilities, dedicated to
studying trace elements in frozen-hydrated biological systems. The BNP is installed at an undulator beamline at Life
Sciences Collaboration Access Team at the Advanced Photon Source. It provides a spatial resolution of 30 nm for
fluorescence imaging by using Fresnel zone plates as nanofocusing optics. Differential phase contrast imaging is
carried out in parallel to fluorescence imaging by using a quadrant photodiode mounted downstream of the sample.
By employing a liquid-nitrogen-cooled sample stage and cryo specimen transfer mechanism, the samples are well
maintained below 110 K during both transfer and X-ray imaging. The BNP is capable for automated tomographic
dataset collection, which enables visualization of internal structures and composition of samples in a nondestructive
manner. In this presentation, we will describe the instrument design principles, quantify instrument performance,
and report the early results that were obtained from frozen-hydrated whole cells.
Near-edge x-ray absorption resonances provide information on molecular orbital structure; these resonances can be exploited in x-ray spectromicroscopy to give sub-50-nanometer resolution images with chemical state sensitivity. At the same time, radiation damage sets a limit to the resolution that can be obtained in absorption mode. Phase contrast imaging may provide another means of chemical state imaging with lower radiation dose. We describe here the use of experimentally measured near-edge absorption data to estimate near-edge phase resonances. This is accomplished by splicing the near-edge data into reference data and carrying out a numerical integration of the Kramers-Kronig relation.
Soft x-ray scanning transmission x-ray microscopy allows one to image dry and wet environmental science, biological, polymer, and geochemical specimens on a nanoscale. Recent advances in instrumentation at the X-1A beamline at the National Synchrotron Light Source at Brookhaven National Laboratory are described. Recent results on Nomarski differential phase contrast and first results on investigations at the oxygen K edge and iron L edge of hydrous ferric oxide transformations are presented.
An integrating solid state detector with segmentation has been developed that addresses the needs in scanning transmission x-ray microscopy below 1 keV photon energy. The detector is not cooled and can be operated without an entrance window which leads to a total photon detection efficiency close to 100%. The chosen segmentation with 8 independent segments is matched to the geometry of the STXM to maximize image mode flexibility. In the bright field configuration for 1 ms integration time and 520 eV x-rays the rms noise is 8 photons per integration.
X-ray microscopy is often discussed as one of the experiments that would benefit from the development of x-ray free-electron lasers. We outline the source characteristics required for several different x-ray microscopy experiments, including possible approaches towards atomic resolution imaging. X-ray FELs would help many but not all of these experiments; those that would benefit the most include experiments that are extremely demanding of a high total flux of coherent x rays; experiments that require snapshot imaging at nanosecond or faster timescales; and pump-probe experiments that require synchronization of an x-ray and a UV laser pulse. In all of these experiments, caution must be taken with regard to radiation and thermal damage.
Scanning transmission x-ray microscopes (STXM) are well matched to the optics of high resolution monochromators, offer a variety of imaging modes and can minimize radiation damage to the specimen. We describe the Stony Brook STXM at the NSLS. This microscope is used for a variety of studies by many users; we briefly outline its use for studies of hydrated colloidal system and for dark field microscopy on immunogold labeled specimens as examples. In order to keep pace with developments in zone plate optics, spectroscopy and a variety of imaging modalities, the microscope is being redesigned and its characteristics are discussed. Its preliminary x-ray detector will be a new multiware proportional counter with high count rate capability.
Barry Winn, X. Hao, Chris Jacobsen, Janos Kirz, J. Miao, Sue Wirick, Harald Ade, Christopher Buckley, Malcolm Howells, Steven Hulbert, Ian McNulty, Tom Oversluizen
The X-1A soft x-ray undulator at the NSLS is the source for our experimental programs in spectromicroscopy. We require both spatial and temporal coherence. Due to the relatively large horizontal divergence of the electron beam in the low (beta) straight section of the x-ray storage ring, it has been possible to split the beam using a scraping mirror into two branches: X-1A used by our program and X-1B used for high resolution spectroscopy. We are now rebuilding the X-1A beamline to provide improved resolving power and essentially linear trade-off between photon rate at the zone plate and resolving power for the soft x-ray spectromicroscopy experiments. This new beamline will exploit both additional floorspace due to the NSLS building expansion and increases in the brightness of the x-ray ring. Our beam will be further split into two separate beamlines, both of which will use toroidal mirrors to focus the source on the monochromator entrance slits horizontally and to focus on the monochromator exit slits vertically. This separation comes at no loss of coherent flux and permits low thermal loading on the optics, since we need little more than the coherent fraction of the beam at the Fresnel zone plate for microfocusing. Because of the small angular acceptance for spatially coherent illumination of the zone plates and the use of an approximately satisfied Rowland condition, our monochromators have sufficient resolving power with fixed exit arms. Experiments can then be placed near the exit slits, with spatial coherence established by the exit slit size. Resolving power will be controlled by adjusting the entrance slit alone with no change of spatial coherence. The zone plates will be overfilled to be less sensitive to beam vibration and drift.
Jorg Maser, Henry Chapman, Chris Jacobsen, Alex Kalinovsky, Janos Kirz, Angelika Osanna, Steve Spector, Steve Wang, Barry Winn, Sue Wirick, Xiaodong Zhang
The Stony Brook scanning transmission x-ray microscope (STXM) has been operating at the X1A beamline at the NSLS since 1989. A large number of users have used it to study biological and material science samples. We report on changes that have been performed in the past year, and present recent results. To stabilize the position of the micro probe when doing spectral scans at high spatial resolution, we have constructed a piezo-driven flexure stage which carries out the focusing motion of the zone plate needed when changing the wavelength. To overcome our detector limitation set by saturation of our gas-flow counter at count rates around 1 MHz, we are installing an avalanche photo diode with an active quenching circuit which we expect to respond linearly to count rates in excess of 10 MHz. We have improved the enclosure for STXM to improve the stability of the Helium atmosphere while taking data. This reduces fluctuations of beam absorption and, therefore, noise in the image. A fast shutter has been installed in the beam line. We are also developing a cryo- STXM which is designed for imaging frozen hydrated samples at temperatures below 120 K. At low temperatures, radiation sensitive samples can tolerate a considerably higher radiation dose than at room temperature. This should improve the resolution obtainable from biological samples and should make recording of multiple images of the same sample area possible while minimizing the effects of radiation damage. This should enable us to perform elemental and chemical mapping at high resolution, and to record the large number of views needed for 3D reconstruction of the object.
Certain types of x-ray microscopes require coherent beams of soft x-rays, and thus are amongst the most brightness-hungry of synchrotron radiation experiments. We consider here what advances third generation sources will and will not offer to several kinds of x-ray microscopes.
Imaging with soft x rays having energies between the carbon and oxygen K edge (284 - 531 eV) yields large absorption contrast for wet organic specimens, but these soft x rays are known to be very effective in damaging biological specimens. The commonly used criterion of mass loss was employed for assessing radiation damage in the scanning transmission x-ray microscope. Multiple images of freeze-dried V. faba chromosomes show no significant mass loss after 150 Mrad. Experiments performed on fixed hydrated chromosomes revealed them to be radiation sensitive. The greater total mass loss observed in multiple low dose images compared to that incurred during a single high dose image suggests that the effects of radiation damage occur slower than the acquisition time for neighboring pixels. The radiation sensitivity of chromosomes depends critically on the fixative used, with damage minimized in glutaraldehyde fixed samples. Radiation damage to chromosomes is independent of ionic strength above 65 mM, but increases for ionic strengths below 65 mM. Using free radical scavengers in the buffer, and changing the design of the sample cell reduced the amount of damage incurred as a function of dose.
Interactions of x rays with a sample are studied to determine the optimal wavelength, source energy, and exposure time for microcopy and holography. The optimal wavelength is influenced by two criteria: minimizing the required source energy and minimizing the absorbed dose and subsequent damage to the sample. The use of heavy element labels, such as colloidal gold, can significantly reduce the energy and dose. Limits to the exposure time due to natural motions, x-ray induced chemical damage, heat build-up, and hydrodynamic expansion are discussed.
Scanning luminescence x-ray microscopy is based on collecting visible light emission from a sample region illuminated by an x-ray microprobe. We have tested the resolution of the method using P31 phosphor grains, and have obtained luminescence images of dye-labelled polystyrene spheres and sodium salicylate crystals. However, we have observed no light emission from the dyes DAPI, ethidium bromide, Hoecst 33258, and rhodamine phalloidon. Present efforts are aimed at improving our understanding of the luminescence process so as to find appropriate dyes for imaging dye-labelled biological specimens.
Several groups have been developing x-ray microscopes for studies of biological and materials specimens at suboptical resolution. The X1A scanning transmission x-ray microscope at Brookhaven National Laboratory has achieved 55 nm Rayleigh resolution, and is limited by the 45 nm finest zone width of the zone plate used to focus the x rays. In principle, features as small as half the outermost zone width, or 23 nm, can be observed in the microscope, though with reduced contrast in the image. One approach to recover the object from the image is to deconvolve the image with the point spread function (PSF) of the optic system. Toward this end, the magnitude of the Fourier transform of the PSF, the modulation transfer function, has been experimentally determined and agrees reasonably well with the calculations using the known parameters of the microscope. To minimize artifacts in the deconvolved images, large signal to noise ratios are required in the original image, and high frequency filters can be used to reduce the noise at the expense of resolution. In this way we are able to recover the original contrast of high resolution features in our images.
Recent work in Gabor x-ray holography has had a resolution limit imposed by the method used to extract the hologram information form the photoresist recording medium. In our case, we believe spiral distortions in the transmission electron microscope used for hologram readout limit resolution to 56 nm. To overcome this limitation we are building a scanning force microscope with a linear scanning stage offering < 20 nm resolution over a (70 micrometers X 70 micrometers ) field and a field linearity of 1 part in 10,000. A field linearity yielding one half or less pixel registration error across the scan is desirable so that the resulting hologram reconstruction is not significantly degraded. This desire for a large and linear scanning field necessitated designing our own stage since these conditions could not be met commercially. It is our goal to use this microscope to achieve higher resolution in reconstructed holograms. In addition, it should offer a means to explore at a macromolecular level the resolution limit of resists, such as PMMA. In this report we describe the technical strategy employed to meet these specifications.
Tomographic x-ray holography may make possible the imaging of biological objects at high resolution in three dimensions. We performed a demonstration experiment with soft x-rays to explore the feasibility of this technique. Coherent 3.2 nm undulator radiation was used to record Fourier transform holograms of a microfabricated test object from various illumination angles. The holograms were numerically reconstructed according to the principles of diffraction tomography, yielding images of the object that are well resolved in three dimensions.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.