X-ray microtomosynthesis of unstained pathology tissue samples.

In pathology protocols, a tissue block, such as one containing a mouse brain or a biopsy sample from a patient, can produce several hundred thin sections. Substantial time may be required to analyse all sections. In cases of uncertainty regarding which sections to focus on, noninvasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sub-10 µm resolution. We explored a novel x-ray tomosynthesis method as a way to maximise contrast-to-noise ratio, a determinant of tissue visibility. It provided a z-stack of thousands of images at 7.3 µm resolution (10% contrast, half-period of 68.5 line pairs/mm), in scans of 5-15 minutes. When compared with micro-CT scans, the straight-line tomosynthesis scan did not need to rotate the sample, which allowed flat samples, such as paraffin blocks, to be kept as close as possible to the x-ray source. Thus, given the same hardware, scan time and resolution, this mode maximised the photon flux density through the sample, which helped in maximising the contrast-to-noise ratio. The tradeoff of tomosynthesis is incomplete 3D information. The microtomosynthesis scanner has scanned 110 unstained human and animal tissue samples as part of their respective pathology protocols. In all cases, the z-stack of images showed tissue structures that guided sectioning or provided correlative structural information. We describe six examples that presented different levels of visibility of soft tissue structures. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.

A microscopy version of the imaging method for 3D luggage screening has been adapted to image unstained pathology samples. Pathology tests of tissue samples are used for clinical diagnosis and for biomedical research. The tissue samples are often embedded in paraffin blocks and sectioned into many thin slices, which are then stained with the appropriate agents for light microscopy. Since each tissue block can produce several hundred thin sections, much time and labour is required to analyse all sections. Noninvasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sufficient resolution. X-ray imaging is a promising tool to meet the challenge since x-rays can penetrate thick samples that are opaque to visible light. With x-ray imaging, a determinant of tissue visibility is the flux density of photons that illuminate the sample. We explored a novel x-ray tomosynthesis method as a way to maximise this factor. It provided a stack of thousands of cross-sectional images at 7.3 µm resolution (half-period of 68.5 line pairs/mm) in scans of 5-15 minutes. When compared with micro-CT scans (a widely used laboratory technology), this method did not need to rotate the sample, which allowed flat samples such as paraffin blocks to be kept as close as possible to the x-ray source. Thus, given the same hardware, scan time and resolution, this method maximised the photon flux density through the sample, which helped in improving the visibility of unstained tissue under x-ray. The tradeoff of the method is incomplete 3D information. Over 100 unstained human and animal tissue samples have been scanned with this method as part of their respective pathology protocols. In all cases, the stack of cross-sectional images showed tissue structures that guided pathology analysis or provided correlative structural information. We describe six examples that presented different levels of tissue visibility. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.

Motionless phase stepping in X-ray phase contrast imaging with a compact source.

X-ray phase contrast imaging offers a way to visualize the internal structures of an object without the need to deposit significant radiation, and thereby alleviate the main concern in X-ray diagnostic imaging procedures today. Grating-based differential phase contrast imaging techniques are compatible with compact X-ray sources, which is a key requirement for the majority of clinical X-ray modalities. However, these methods are substantially limited by the need for mechanical phase stepping. We describe an electromagnetic phase-stepping method that eliminates mechanical motion, thus removing the constraints in speed, accuracy, and flexibility. The method is broadly applicable to both projection and tomography imaging modes. The transition from mechanical to electromagnetic scanning should greatly facilitate the translation of X-ray phase contrast techniques into mainstream applications.

Online Calibration of a Linear Micro Tomosynthesis Scanner.

In a linear tomosynthesis scanner designed for imaging histologic samples of several centimeters size at 10 µm resolution, the mechanical instability of the scanning stage (±10 µm) exceeded the resolution of the image system, making it necessary to determine the trajectory of the stage for each scan to avoid blurring and artifacts in the images that would arise from the errors in the geometric information used in 3D reconstruction. We present a method for online calibration by attaching a layer of randomly dispersed micro glass beads or calcium particles to the bottom of the sample stage. The method was based on a parametric representation of the rigid body motion of the sample stage-marker layer assembly. The marker layer was easy to produce and proven effective in the calibration procedure.

Online Geometric Calibration of a Hybrid CT System for Ultrahigh-Resolution Imaging.

A hybrid imaging system consisting of a standard computed tomography (CT) scanner and a low-profile photon-counting detector insert in contact with the patient's body has been used to produce ultrahigh-resolution images in a limited volume in chest scans of patients. The detector insert is placed on the patient bed as needed and not attached. Thus, its position and orientation in the scanner is dependent on the patient's position and scan settings. To allow accurate image reconstruction, we devised a method of determining the relative geometry of the detector insert and the CT scanner for each scan using fiducial markers. This method uses an iterative registration algorithm to align the markers in the reconstructed volume from the detector insert to that of the concurrent CT scan. After obtaining precise geometric information of the detector insert relative to the CT scanner, the two complementary sets of images are summed together to create a detailed image with reduced artifacts.

Theory of oblique and grazing incidence Talbot­Lau interferometers and demonstration in a compact source x­ray reflective interferometer.

With the advent of Talbot-Lau interferometers for x-ray phase-contrast imaging, oblique and grazing incidence configurations are now used in the pursuit of sub-micron grating periods and high sensitivity. Here we address the question whether interferometers having oblique incident beams behave in the same way as the well-understood normal incidence ones, particularly when the grating planes are non-parallel. We derive the normal incidence equivalence of oblique incidence geometries from wave propagation modeling. Based on the theory, we propose a practical method to correct for non-parallelism of the grating planes, and demonstrate its effectiveness with a polychromatic hard x-ray reflective interferometer.

Interpretation of dark-field contrast and particle-size selectivity in grating interferometers.

In grating-based x-ray phase sensitive imaging, dark-field contrast refers to the extinction of the interference fringes due to small-angle scattering. For configurations where the sample is placed before the beamsplitter grating, the dark-field contrast has been quantified with theoretical wave propagation models. Yet when the grating is placed before the sample, the dark-field contrast has only been modeled in the geometric optics regime. Here we attempt to quantify the dark-field effect in the grating-before-sample geometry with first-principle wave calculations and understand the associated particle-size selectivity. We obtain an expression for the dark-field effect in terms of the sample material's complex refractive index, which can be verified experimentally without fitting parameters. A dark-field computed tomography experiment shows that the particle-size selectivity can be used to differentiate materials of identical x-ray absorption.

Simultaneous myocardial strain and dark-blood perfusion imaging using a displacement-encoded MRI pulse sequence.

The purpose of this study is to develop and evaluate a displacement-encoded pulse sequence for simultaneous perfusion and strain imaging. Displacement-encoded images in two to three myocardial slices were repeatedly acquired using a single-shot pulse sequence for 3 to 4 min, which covers a bolus infusion of Gadolinium contrast. The magnitudes of the images were T(1) weighted and provided quantitative measures of perfusion, while the phase maps yielded strain measurements. In an acute coronary occlusion swine protocol (n = 9), segmental perfusion measurements were validated against microsphere reference standard with a linear regression (slope 0.986, R(2) = 0.765, Bland-Altman standard deviation = 0.15 mL/min/g). In a group of ST-elevation myocardial infarction patients (n = 11), the scan success rate was 76%. Short-term contrast washout rate and perfusion are highly correlated (R(2) = 0.72), and the pixelwise relationship between circumferential strain and perfusion was better described with a sigmoidal Hill curve than linear functions. This study demonstrates the feasibility of measuring strain and perfusion from a single set of images.

Selective imaging of nano-particle contrast agents by a single-shot x-ray diffraction technique.

Iron oxide nano-particles have very different x-ray diffraction properties from tissue. They can be clearly visualized against suppressed tissue background in a single-shot x-ray diffraction imaging technique. This technique is able to acquire both diffraction and absorption images from a single grating-modulated projection image through analysis in the spatial frequency domain. We describe the use of two orthogonal transmission gratings to selectively retain diffraction signal from iron oxide particles that are larger than a threshold size, while eliminating the background signal from soft tissue and bone. This approach should help the tracking of functionalized particles in cell labeling and targeted therapy.

Grazing angle Mach-Zehnder interferometer using reflective phase gratings and a polychromatic, un-collimated light source.

Normal incidence Talbot-Lau interferometers in x-ray applications have the drawbacks of low fringe visibility with polychromatic sources when the wave propagation distance is increased to achieve higher phase sensitivity, and when fabrication limits the attainable grating density. In contrast, reflective gratings illuminated at grazing angles have dramatically higher effective densities than their physical values. However, new designs are needed for far field interferometers using grazing angle geometry with incoherent light sources. We show that, with the appropriate design and choice of reflective phase gratings, there exist pairs of interfering pathways of exactly equal lengths independent of the incoming beam's incidence angle and wavelength. With a visible light grazing angle Mach-Zehnder interferometer, we show the conditions for achieving near ideal fringe visibility and demonstrate both absolute and differential phase-contrast imaging. We also describe the design parameters of an x-ray interferometer and key factors for its implementation.

Single-shot x-ray differential phase-contrast and diffraction imaging using two-dimensional transmission gratings.

We describe an x-ray differential phase-contrast imaging method based on two-dimensional transmission gratings that are directly resolved by an x-ray camera. X-ray refraction and diffraction in the sample lead to variations of the positions and amplitudes of the grating fringes on the camera. These effects can be quantified through spatial harmonic analysis. The use of 2D gratings allows differential phase contrast in several directions to be obtained from a single image. When compared to previous grating-based interferometry methods, this approach obviates the need for multiple exposures and separate measurements for different directions and thereby accelerates imaging speed.

A grating-based single-shot x-ray phase contrast and diffraction method for in vivo imaging.

PURPOSE: The purpose of this study is to develop a single-shot version of the grating-based phase contrast x-ray imaging method and demonstrate its capability of in vivo animal imaging. Here, the authors describe the principle and experimental results. They show the source of artifacts in the phase contrast signal and optimal designs that minimize them. They also discuss its current limitations and ways to overcome them. METHODS: A single lead grid was inserted midway between an x-ray tube and an x-ray camera in the planar radiography setting. The grid acted as a transmission grating and cast periodic dark fringes on the camera. The camera had sufficient spatial resolution to resolve the fringes. Refraction and diffraction in the imaged object manifested as position shifts and amplitude attenuation of the fringes, respectively. In order to quantify these changes precisely without imposing a fixed geometric relationship between the camera pixel array and the fringes, a spatial harmonic method in the Fourier domain was developed. The level of the differential phase (refraction) contrast as a function of hardware specifications and device geometry was derived and used to guide the optimal placement of the grid and object. Both ex vivo and in vivo images of rodent extremities were collected to demonstrate the capability of the method. The exposure time using a 50 W tube was 28 s. RESULTS: Differential phase contrast images of glass beads acquired at various grid and object positions confirmed theoretical predictions of how phase contrast and extraneous artifacts vary with the device geometry. In anesthetized rats, a single exposure yielded artifact-free images of absorption, differential phase contrast, and diffraction. Differential phase contrast was strongest at bone-soft tissue interfaces, while diffraction was strongest in bone. CONCLUSIONS: The spatial harmonic method allowed us to obtain absorption, differential phase contrast, and diffraction images, all from a single raw image and is feasible in live animals. Because the sensitivity of the method scales with the density of the gratings, custom microfabricated gratings should be superior to off-the-shelf lead grids.

Fourier X-ray scattering radiography yields bone structural information.

PURPOSE: To characterize certain aspects of the microscopic structures of cortical and trabecular bone by using Fourier x-ray scattering imaging. MATERIALS AND METHODS: Protocols approved by the National Institutes of Health Animal Care and Use Committee were used to examine ex vivo the hind limb of a rat and the toe of a pig. The Fourier x-ray scattering imaging technique involves the use of a grid mask to modulate the cone beam and Fourier spectral filters to isolate the harmonic images. The technique yields attenuation, scattering, and phase-contrast (PC) images from a single exposure. In the rat tibia cortical bone, the scattering signals from two orthogonal grid orientations were compared by using Wilcoxon signed rank tests. In the pig toe, the heterogeneity of scattering and PC signals was compared between trabecular and compact bone regions of uniform attenuation by using F tests. RESULTS: In cortical bone, the scattering signal was significantly higher (P < 10(-15)) when the grid was parallel to the periosteal surface. Trabecular bone, as compared with cortical bone, appeared highly heterogeneous on the scattering (P < 10(-34)) and PC (P < 10(-27)) images. CONCLUSION: The ordered alignment of the mineralized collagen fibrils in compact bone was reflected in the anisotropic scattering signal in this bone. In trabecular bone, the porosity of the mineralized matrix accounted for the granular pattern seen on the scattering and PC images.

Regional Ultrahigh-Resolution Rescan in a Clinical Whole-Body CT Scanner Using a Contact Detector Insert.

Ultrahigh-resolution, low-dose rescans in a region of interest following a general screening computed tomography (CT) scan is motivated by the need to reduce invasive tissue biopsy procedures in cancer screening. We describe a new method to meet the conflicting demands of ultrahigh resolution, high-speed and ultralow-dose, and the first proof-of-concept experiment. With improving detector resolution, the limiting factor for the system resolution of whole-body CT scanners shifts to the penumbra of the source focal spot. The penumbra unsharpness is minimized by inserting flat-panel detector(s) that are in direct contact with the body. In the hybrid system, the detector insert and the CT detector acquire data simultaneously, whereby the standard CT images give the position and orientation of the detector insert(s) as needed for tomosynthesis reconstruction. Imaging tests were performed with a compact photon-counting detector insert on resolution targets of both high- and low-contrast as well as a mouse specimen, all inside a body phantom. Detector insert tomosynthesis provided twice the resolution of the CT scanner alone at the same dose concentration. The short 2-cm beam collimation of the tomosynthesis rescan gave an effective dose equivalent to 6% of an average CT scan in the chest or abdomen.

Adaptive postprocessing techniques for myocardial tissue tracking with displacement-encoded MR imaging.

UNLABELLED: The purpose of this study was to prospectively assess the effects of two adaptive postprocessing techniques on the evaluation of myocardial function with displacement-encoded magnetic resonance (MR) imaging, including sensitivity for abnormal wall motion, with two-dimensional echocardiography as the reference standard. Sixteen patients (11 men, five women; age range, 26-74 years) and 12 volunteers (six men, six women; age range, 29-53 years) underwent breath-hold MR imaging. Institutional review board approval and informed consent were obtained. Adaptive phase-unwrapping and spatial filtering techniques were compared with conventional phase-unwrapping and spatial filtering techniques. Use of the adaptive techniques led to a reduced rate of failure with the phase-unwrapping technique from 18.9% to 0.6% (P < .001), resulted in lower variability of segmental strain measurements among healthy volunteers (P < .001 to P = .02), and increased the sensitivity of quantitative detection of abnormal segments in patients from 82.5% to 87.7% (P = .034). The adaptive techniques improved the semiautomated postprocessing of displacement-encoded cardiac images and increased the sensitivity of detection of abnormal wall motion in patients. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/246/1/229/DC1.

Nano-printed miniature compound refractive lens for desktop hard x-ray microscopy.

Hard x-ray lenses are useful elements in x-ray microscopy and in creating focused illumination for analytical applications such as x-ray fluorescence imaging. Recently, polymer compound refractive lenses for focused illumination in the soft x-ray regime (< 10 keV) have been created with nano-printing. However, there are no such lenses yet for hard x-rays, particularly of short focal lengths for benchtop microscopy. We report the first instance of a nano-printed lens for hard x-ray microscopy, and evaluate its imaging performance. The lens consists of a spherically focusing compound refractive lens designed for 22 keV photon energy, with a tightly packed structure to provide a short total length of 1.8 mm and a focal length of 21.5 mm. The resulting lens technology was found to enable benchtop microscopy at 74x magnification and 1.1 µm de-magnified image pixel size at the object plane. It was used to image and evaluate the focal spots of tungsten-anode micro-focus x-ray sources. The overall system resolution with broadband illumination from a tungsten-anode x-ray tube at 30 kV and 10 mm focal distance was measured to be 2.30±0.22 µm.

Correlative Detection of Isolated Single and Multi-Cellular Calcifications in the Internal Elastic Lamina of Human Coronary Artery Samples.

Histopathology protocols often require sectioning and processing of numerous microscopy slides to survey a sample. Trade-offs between workload and sampling density means that small features can be missed. Aiming to reduce the workload of routine histology protocols and the concern over missed pathology in skipped sections, we developed a prototype x-ray tomographic scanner dedicated to rapid scouting and identification of regions of interest in pathology specimens, thereby allowing targeted histopathology analysis to replace blanket searches. In coronary artery samples of a deceased HIV patient, the scanner, called Tomopath, obtained depth-resolved cross-sectional images at 15 µm resolution in a 15-minute scan, which guided the subsequent histological sectioning and microscopy. When compared to a commercial tabletop micro-CT scanner, the prototype provided several-fold contrast-to-noise ratio in 1/11th the scan time. Correlated tomographic and histological images revealed two types of micro calcifications: scattered loose calcifications typically found in atherosclerotic lesions; isolated focal calcifications in one or several cells in the internal elastic lamina and occasionally in the tunica media, which we speculate were the initiation of medial calcification linked to kidney disease, but rarely detected at this early stage due to their similarity to particle contaminants introduced during histological processing, if not for the evidence from the tomography scan prior to sectioning. Thus, in addition to its utility as a scouting tool, in this study it provided complementary information to histological microscopy. Overall, the prototype scanner represents a step toward a dedicated scouting and complementary imaging tool for routine use in pathology labs.

Geometric calibration and correction for a lens-coupled detector in x-ray phase-contrast imaging.

A lens-coupled x-ray camera with a tilted phosphor collects light emission from the x-ray illuminated (front) side of phosphor. Experimentally, it has been shown to double x-ray photon capture efficiency and triple the spatial resolution along the phosphor tilt direction relative to the same detector at normal phosphor incidence. These characteristics benefit grating-based phase-contrast methods, where linear interference fringes need to be clearly resolved. However, both the shallow incident angle on the phosphor and lens aberrations of the camera cause geometric distortions. When tiling multiple images of limited vertical view into a full-field image, geometric distortion causes blurring due to image misregistration. Here, we report a procedure of geometric correction based on global polynomial transformation of image coordinates. The corrected image is equivalent to one obtained with a single full-field flat panel detector placed at the sample plane. In a separate evaluation scan, the position deviations in the horizontal and vertical directions were reduced from 0.76 and 0.028 mm, respectively, to 0.006 and 0.009 mm, respectively, by the correction procedure, which were below the 0.028-mm pixel size of the imaging system. In a demonstration of a phase-contrast imaging experiment, the correction reduced blurring of small structures.

A Universal Moiré Effect and Application in X-Ray Phase-Contrast Imaging.

A moiré pattern is created by superimposing two black-and-white or gray-scale patterns of regular geometry, such as two sets of evenly spaced lines. We observed an analogous effect between two transparent phase masks in a light beam which occurs at a distance. This phase moiré effect and the classic moiré effect are shown to be the two ends of a continuous spectrum. The phase moiré effect allows the detection of sub-resolution intensity or phase patterns with a transparent screen. When applied to x-ray imaging, it enables a polychromatic far-field interferometer (PFI) without absorption gratings. X-ray interferometry can non-invasively detect refractive index variations inside an object1-10. Current bench-top interferometers operate in the near field with limitations in sensitivity and x-ray dose efficiency2, 5, 7-10. The universal moiré effect helps overcome these limitations and obviates the need to make hard x-ray absorption gratings of sub-micron periods.

Electrodeposition of Gold to Conformally Fill High Aspect Ratio Nanometric Silicon Grating Trenches: A Comparison of Pulsed and Direct Current Protocols.

Filling high-aspect-ratio trenches with gold is a frequent requirement in the fabrication of x-ray optics as well as micro-electronic components and other fabrication processes. Conformal electrodeposition of gold in sub-micron-width silicon trenches with an aspect ratio greater than 35 over a grating area of several square centimeters is challenging and has not been described in the literature previously. A comparison of pulsed plating and constant current plating led to a gold electroplating protocol that reliably filled trenches for such structures.

Enhancing Tabletop X-Ray Phase Contrast Imaging with Nano-Fabrication.

X-ray phase-contrast imaging is a promising approach for improving soft-tissue contrast and lowering radiation dose in biomedical applications. While current tabletop imaging systems adapt to common x-ray tubes and large-area detectors by employing absorptive elements such as absorption gratings or monolithic crystals to filter the beam, we developed nanometric phase gratings which enable tabletop x-ray far-field interferometry with only phase-shifting elements, leading to a substantial enhancement in the performance of phase contrast imaging. In a general sense the method transfers the demands on the spatial coherence of the x-ray source and the detector resolution to the feature size of x-ray phase masks. We demonstrate its capabilities in hard x-ray imaging experiments at a fraction of clinical dose levels and present comparisons with the existing Talbot-Lau interferometer and with conventional digital radiography.