ABSTRACT
We demonstrate a modal approach to simultaneous recovery of a sample transmission function and coherence properties of an illuminating X-ray beam that works from a single measurement. The approach based on coherence diffractive imaging, does not depend on a particular model for the coherence function. This single shot imaging method separates the effects of partial coherence in the illuminating beam from the sample, allowing the recovery of high quality sample information.
ABSTRACT
Recent developments in biological X-ray microscopy have allowed structural information and elemental distribution to be simultaneously obtained by combining X-ray ptychography and X-ray fluorescence microscopy. Experimentally, these methods can be performed simultaneously; however, the optimal conditions for each measurement may not be compatible. Here, we combine two distinct measurements of ultrastructure and elemental distribution, with each measurement performed under optimised conditions. By combining optimised ptychography and fluorescence information we are able to determine molar concentrations from two-dimensional images, allowing an investigation into the interactions between the environment sensing filopodia in fibroblasts and extracellular calcium. Furthermore, the biological ptychography results we present illustrate a point of maturity where the technique can be applied to solve significant problems in structural biology.
Subject(s)
Elements , Extracellular Matrix/chemistry , Fibroblasts/ultrastructure , Image Processing, Computer-Assisted/methods , Microscopy/methods , Spectrometry, X-Ray Emission/methods , X-Ray Diffraction/methods , Animals , Cells, Cultured , MiceABSTRACT
We describe a comprehensive model of a commercial indirect X-ray imaging detector that accurately predicts the detector point spread function and its dependence on X-ray energy. The model was validated by measurements using monochromatic synchrotron radiation and extended to polychromatic X-ray sources. Our approach can be used to predict the performance of an imaging detector and can be used to optimize imaging experiments with broad-band X-ray sources.
ABSTRACT
We demonstrate the application of a complex constraint in the reconstruction of images from phase-diverse Fresnel coherent diffraction data for heterogeneous biological objects. The application of this constraint is shown to improve the quality of the reconstruction of both the phase and the magnitude of the complex object transmission function.
Subject(s)
Algorithms , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Microscopy, Phase-Contrast/methods , Refractometry/methodsABSTRACT
Phase-diverse Fresnel coherent diffractive imaging has been shown to reveal the structure and composition of biological specimens with high sensitivity at nanoscale resolution. However, the method has yet to be applied using X-ray illumination with energy in the so-called 'water-window' that lies between the carbon and oxygen K edges. In this range, differences in the strength of the X-ray interaction for protein based biological materials and water is increased. Here we demonstrate a proof-of-principle application of FCDI at an X-ray energy within the water-window to a dehydrated cellular sample composed of red blood cells infected with the trophozoite stage of the malaria parasite, Plasmodium falciparum. Comparison of the results to both optical and electron microscopy shows that the correlative imaging methods that include water-window FCDI will find utility in studying cellular architecture.
Subject(s)
Erythrocytes/parasitology , Erythrocytes/ultrastructure , Image Enhancement/methods , Malaria, Falciparum/pathology , Malaria/pathology , Refractometry/methods , X-Ray Diffraction/methods , Malaria/diagnostic imaging , Malaria/parasitology , Malaria, Falciparum/diagnostic imaging , Malaria, Falciparum/parasitology , Microscopy, Phase-Contrast/methods , Radiography , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We demonstrate Fresnel Coherent Diffractive Imaging (FCDI) tomography in the X-ray regime. The method uses an incident X-ray illumination with known curvature in combination with ptychography to overcome existing problems in diffraction imaging. The resulting tomographic reconstruction represents a 3D map of the specimen's complex refractive index at nano-scale resolution. We use this technique to image a lithographically fabricated glass capillary, in which features down to 70nm are clearly resolved.
ABSTRACT
A dedicated in-vacuum coherent x-ray diffraction microscope was installed at the 2-ID-B beamline of the Advanced Photon Source for use with 0.7-2.9 keV x-rays. The instrument can accommodate three common implementations of diffractive imaging; plane wave illumination; defocused-probe (Fresnel diffractive imaging) and scanning (ptychography) using either a pinhole, focused or defocused probe. The microscope design includes active feedback to limit motion of the optics with respect to the sample. Upper bounds on the relative optics-to-sample displacement have been measured to be 5.8 nm(v) and 4.4 nm(h) rms/h using capacitance micrometry and 27 nm/h using x-ray point projection imaging. The stability of the measurement platform and in-vacuum operation allows for long exposure times, high signal-to-noise and large dynamic range two-dimensional intensity measurements to be acquired. Finally, we illustrate the microscope's stability with a recent experimental result.
