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.
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.
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.
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.
The requirements on the spatial and temporal coherence for conventional Coherent Diffractive Imaging
(CDI) have been well-established in the literature based on Shannon sampling of the diffracted intensities. The
spatial coherence length of the illumination must be larger than twice the lateral dimensions of the sample whilst the
temporal coherence length must be larger than the maximum optical path length difference between the two edges of
the sample for the highest order diffraction peaks. However, recent approaches to CDI which have included
knowledge of the spatial and temporal coherence information in the image reconstruction have allowed us to relax
these conventional coherence constraints, extending the applicability of the technique to less coherent sources. In
light of these developments it is useful to revisit the idea of a coherence limit in partially coherent CDI and establish
a ‘universal’ limit on the partial coherence that can be tolerated without any loss of information. In this paper we
present a simple and straightforward description of the limit of spatial and temporal coherence in partially coherent