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.
Zone plates are diffractive focusing optics capable of nanometer focusing but limited focusing efficiency at hard x-ray energy. A smaller focus spot is possible by reducing the outer zone width (OZW) while increasing the zone height will generally increase focusing efficiency. The combination of thick zones with small outer zone width, or high aspect ratio, for better performing zone plates is not feasible with state-of-the-art fabrication methods and requires other methods to achieve the aspect ratio desired. Near-field stacking involves two zone plates with the same dimensions and aligning them within the depth of focus in the beam direction and one third of the OZW in the transverse direction. Due to the depth of focus limitation, stacking more than 2 zone plates is extremely difficult, so a new method was proposed and developed to stack zone plates in the intermediate field. Multiple stacking apparatuses were assembled and tested. We will report on results from stacking 80-nm OZW zone plates from a near-field stacking experiment at 10 keV X-ray energy and intermediate field stacking 6 zone plates at 27 keV X-ray energy. We will also present findings on how to combine the stacking techniques.
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.
Nature has often provided inspiration for new smart structures and materials. Recently, we showed a bundle of a few
wood cells are moisture-activated torsional actuators that can reversibly twist multiple revolutions per centimeter of
length. The bundles produce specific torque higher than that produced by electric motors and possess shape memory
twist capabilities. Here we also report that ion diffusion through wood cell walls is a stimuli-responsive phenomenon.
Using the high spatial resolution and sensitivity of synchrotron-based x-ray fluorescence microscopy (XFM), metal ions
deposited into individual wood cell walls were mapped. Then, using a custom-built relative humidity (RH) chamber,
diffusion of the metal ions was observed in situ first at low RH and then at increasingly higher RH. We found that ions
did not diffuse through wood cell walls at low RH, but diffusion occurred at high RH. We propose that both the shape
memory twist effect and the moisture content threshold for ionic diffusion are controlled by the hemicelluloses passing
through a moisture-dependent glass transition in the 60-80% RH range at room temperature. An advantage of wood over
other stimuli-responsive polymers is that wood lacks bulk mechanical softening at the transition that controls the stimuliresponsive
behavior. We demonstrate using a custom-built torque sensor that the torque generation in wood cell bundles
actually continues to increase over the RH range that hemicelluloses soften. The hierarchical structure of wood provides
the inspiration to engineer stimuli-responsive polymers and actuators with increased mechanical strength and higher
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.
Kirkpatrick-Baez (K-B) mirrors  are sophisticated x-ray micro- and nano-focusing tools for synchrotron radiation applications. A prototype of a modular x-ray K-B mirror mount system has been designed and tested at an optics testing beamline, 1-BM at the Advanced Photon Source (APS), Argonne National Laboratory (ANL). This compact, costeffective modular mirror mount system is designed to meet challenging mechanical and optical specifications for producing high positioning resolution and stability for various scientific applications with focused hard x-ray beams down to the 100-nanometer scale. The optomechanical design of the modular x-ray K-B mirror mount system as well as the preliminary test results of its precision positioning performance are presented in this paper.
We have developed a prototype instrument with a novel interferometrically controlled differential scanning stage system. The system consists of 9 DC-motor-driven stages, 4 picomotor-driven stages, and 2 PZT-driven stages. A custom-built laser Doppler displacement meter system provides two-dimensional (2D) differential displacement measurement with subnanometer resolution between the zone-plate x-ray optics and the sample holder. The entire scanning system was designed with high stiffness, high repeatability, low drift, flexible scanning schemes, and possibility of fast feedback for differential motion. Designs of the scanning stage system, as well as preliminary mechanical test results, are presented in this paper.
We introduce a new design of tilted linear zone plates, which are named tapered tilted linear (TTL) zone plates. The purpose of the design is to increase efficiency while at the same time keeping the focal plane perpendicular to the optical path. In order to accomplish this, the zone radius and number of zones must become a function of position along the structure. Simulation work described in this paper shows improved optical performance over regular tilted linear zone plates.
Using Fresnel zone plates, a spatial resolution between 20 nm for soft x-rays and 70 nm for hard x-rays has been achieved. Improvement of the spatial resolution without loss of efficiency is difficult and incremental due to the fabrication challenges posed by the combination of small outermost zone width and high aspect ratios. We describe a novel approach for high-resolution x-ray focusing, a multilayer Laue lens (MLL). The MLL concept is a system of two crossed linear zone plates, manufactured by deposition techniques. The approach involves deposition of a multilayer with a graded period, sectioning it to the appropriate thickness, assembling the sections at the optimum angle, and using it in Laue geometry for focusing. The approach is particularly well suited for high-resolution focusing optics for use at high photon energy. We present a theory of the MLL using dynamic diffraction theory and Fourier optics.
For several years efforts have been made to improve the resolution for imaging and tomography with hard X-rays. Recently we demonstrated sub-100 nm resolution at 13 keV with a microscope including a Kirkpatrick-Baez multilayer-mirror (KB) as a condenser followed by a micro-Fresnel Zone Plate (FZP) as an objective lens. We built since a new tomography station at UNICAT at the Advanced Photon Source integrating the KB-FZP microscope for 100 nm tomography.
By employing the natural absorption contrast of organic matter in water at 0.5 keV photon energy, X-ray microcopy has resolved 30 nm structures in animal cells. To protect the cells from radiation damage caused by x-rays, imaging of the samples was performed at cryogenic temperatures, which makes it possible to take multiple images of a single cell. Due to the small numerical aperture of zone plates, X-ray objectives have a depth of focus on the order of several microns. By treating the X-ray microscopic images as projections of the sample absorption, computed tomography (CT) can be performed. Since cryogenic biological samples are resistant to radiation damage, it is possible to reconstruct frozen-hydrated cells imaged with a full-field X-ray microscope. This approach is used to obtain three- dimensional information about the location of specific proteins in cells. To localize proteins in cells, immunolabelling with strongly X-ray absorbing nanoparticles was performed. With the new tomography apparatus developed for the X-ray microscope XM-1 installed at the ALS, we have performed tomography of immunolabelled frozen-hydrated cells to detect protein distributions in all three dimensions inside of cells. As a first example, the distribution of the nuclear protein, male specific lethal 1 (MSL-1) in the Drosophila melanogaster cell was studied.