Knife-edge Scanning Research Articles

High-resolution terrain imager development and performance evaluation for lunar exploration.

The Lunar Terrain Imager (LUTI) is a crucial scientific instrument aboard the Korea Pathfinder Lunar Orbiter. We present an analysis of the optical design, assembly, and performance of the LUTI camera system. We outline the key components and technical specifications of the cameras and detail the assembly and alignment process, including collimator calibration and the knife-edge scanning technique for modulation transfer function measurements. Furthermore, the thermal vacuum optical performance of the LUTI is evaluated by simulating space conditions. The optical design, assembly, and thermal vacuum performance testing make the system valuable for high-resolution lunar surface imaging and future space exploration missions.

Performance of micro-beam X-ray absorption spectroscopy at beamline 7.2W of Synchrotron Light Research Institute

The existing BL7.2W beamline at SLRI, initially developed for Macromolecular Crystallography, was upgraded with a Si-drift X-ray detector, sample micro-stage, and polycapillary X-ray lens to provide possibilities for experiments using X-ray fluorescence (XRF) imaging and micro-beam X-ray absorption spectroscopy (μXAS) techniques. The micro X-ray beam was characterized. The beam diameter of approximately 25 μm was determined from the knife-edge scan method. The measured photon flux was 3.8 × 109, 1.8 × 109, and 6.4 × 108 photons/s for 8.0, 10.0, and 12.0 keV X-rays, respectively. Imaging XRF and μXAS measurements were successfully performed on a welding joint of oxygen-free high thermal conductivity copper and AISI304 stainless steel. This opens opportunities for SLRI users to utilize this new measurement setup for elemental and chemical characterizations of small areas on various samples.

Investigation of polycapillary half lenses for quantitative confocal micro-X-ray fluorescence analysis.

The use of polycapillary optics in confocal micro-X-ray fluorescence analysis (CMXRF) enables the destruction-free 3D investigation of the elemental composition of samples. The energy-dependent transmission properties, concerning intensity and spatial beam propagation of three polycapillary half lenses, which are vital for the quantitative interpretation of such CMXRF measurements, are investigated in a monochromatic confocal laboratory setup at the Atominstitut of TU Wien, and a synchrotron setup on the BAMline beamline at the BESSY II Synchrotron, Helmholtz-Zentrum-Berlin. The empirically established results, concerning the intensity of the transmitted beam, are compared with theoretical values calculated with the polycap software package and a newly presented analytical model for the transmission function. The resulting form of the newly modelled energy-dependent transmission function is shown to be in good agreement with Monte Carlo simulated results for the complete energy regime, as well as the empirically established results for the energy regime between 6 keV and 20 keV. An analysis of possible fabrication errors was conducted via pinhole scans showing only minor fabrication errors in two of the investigated polycapillary optics. The energy-dependent focal spot size of the primary polycapillary was investigated in the laboratory via the channel-wise evaluation of knife-edge scans. Experimental results are compared with data given by the manufacturer as well as geometric estimations for the minimal focal spot size. Again, the resulting measurement points show a trend in agreement with geometrically estimated results and manufacturer data.

Focus characterization of an X-ray free-electron laser by intensity correlation measurement of X-ray fluorescence.

This paper proposes and demonstrates a simple method using the intensity correlation of X-ray fluorescence to evaluate the focused beam size of an X-ray free-electron laser (XFEL). This method was applied to the sub-micrometre focused XFEL beam at the SPring-8 Angstrom Compact Free Electron Laser, and the beam size evaluated using the proposed method was consistent with that measured using the knife-edge scan method. The proposed method is readily applicable to extremely small X-ray spots and can be applied for the precise diagnostics of sub-10 nm focused X-ray beams which have recently emerged.

CRL optics and silicon drift detector for P06 Microprobe experiments at 35 keV

A provisional setup for X-ray microprobe experiments at 35 keV is described. It is based on compound refractive lenses (CRLs) for nanofocusing and a Vortex silicon drift detector with 2 mm sensor thickness for increased sensitivity at high energies. The Microprobe experiment (PETRA III) generally uses Kirkpatrick-Baez mirrors for submicrometer focusing in the energy range of 5–21 keV. However, various types of scanning X-ray microscopy experiments require higher excitation energies. The CRL optics were characterized by X-ray ptychography and X-ray fluorescence (XRF) knife edge scans on a siemens star pattern and showed beam sizes down to 110 nm. The performance of the new setup for microscopic X-ray diffraction (XRD)–XRF scanning X-ray microscopy measurements at 35 keV is demonstrated on a cross-section of a painting fragment.

MRI size assessment of cerebral microvasculature using diffusion-time-dependent stimulated-echo acquisition: A feasibility study in rodent

In this study, a stimulated-echo (STE) method was employed to robustify the cerebral vessel size estimation near air-tissue, bone-tissue interfaces, and large vessels. The proposed solution is to replace the relaxation rate change from gradient-echo (GRE) with that from STE with long diffusion time after the injection of an intravascular contrast agent, superparamagnetic iron oxide nanoparticles. The corresponding diffusion length of STE is shorter than the length over which the unwanted macroscopic field inhomogeneities but is still longer than the correlation length of the fields induced by small vessels. Therefore, the unwanted field inhomogeneities are refocused, while preserving microscopic susceptibility contrast from cerebral vessels. The mean vessel diameter (dimensionless) derived from the diffusion-time-varying STE method was compared to the mean vessel diameter obtained by a conventional spin-echo (SE) and GRE combination based on Monte-Carlo proton diffusion simulations and in vivo rat experiments at 7 ​T. The in vivo mean vessel diameter from the MRI experiments was directly compared to available reference mouse brain vasculature obtained by a knife-edge scanning microscope (KESM), which is considered to be the gold standard. Monte-Carlo simulation revealed that SE and GRE-based MR relaxation rate changes (ΔR2 and ΔR2∗, respectively) can be enhanced using single STE-based MR relaxation rate change (ΔRSTE) by regulating diffusion time, especially for small vessels. The in vivo mean vessel diameter from the STE method demonstrated a closer agreement with that from the KESM compared to the combined SE and GRE method, especially in the olfactory bulb and cortex. This study demonstrates that STE relaxation rate changes can be used as consistent measures for assessing small cerebral microvasculature, where macroscopic field inhomogeneity is severe and signal contamination from adjacent large vessels is significant.

SPARC: Distribution of Intrinsic Cardiac Neurons in 3D Reconstructed Hearts of F344 Rat

The intrinsic cardiac nervous system (ICN) is involved in the autonomic control of the heart and functions as an integrative center for local sensory and extrinsic autonomic inputs. There has been increasing interest in the functions of ICN neurons as well as their ablation as a therapeutic intervention for atrial fibrillation. The goal of the present study is to assess the distribution of ICN neurons between individuals with rat as a model using a novel data acquisition pipeline combining Knife Edge Scanning Microscope (KESM) with the custom software developed by MBF (Tissue Mapper) to perform single cell mapping of ICN neurons in 3D reconstructed rat hearts. Fischer 344 male rats (n = 3) were perfused through the abdominal aorta and whole‐mount perfusion stained with Cresyl Echt Violet. The hearts were then embedded into paraffin and mounted onto a robotic XYZ stage for simultaneous sectioning and imaging by a KESM. Individual section images that consisted of 1400, 1570, and 2580 sections for each heart that was cut at a 5 μm thickness were assembled into an image stack for anatomical cardiac digitization and single cell mapping of ICN neurons in Tissue Mapper for 3D heart reconstruction. The distribution of ICN neurons between individuals was assessed in two different planes of section: parallel sections showing 4‐chambers, and cross sections perpendicular to the long axis of the heart. The number of neurons counted in each of the heart samples were 2676, 2973, and 2885, with an average of 2844/rat. ICN neurons mainly localized around the posterior surface of the left and right atria and the hilum ‐ pulmonary veins, interatrial septum, along the pulmonary artery, and the root of the superior vena cava. We performed computational analysis of ICN neuron distribution using a partial projection approach based on principal component analysis of the 3D coordinates. We analyzed the first two principal components to compute a distribution of packing density in the 2D projection corresponding to maximum variability in the spatial location of single neurons. In parallel, we conducted unsupervised clustering of 3D coordinates of single neurons using partitioning around medoids algorithm and annotated the packing density distributions and 3D ICN neuron maps with the resultant clusters. Our analysis revealed that the density distribution, numbers of clusters and orientation of ICN neurons were similar across males with 7–10 clusters within each ICN. This demonstrates the robustness of the technique where single ICN neurons were well‐preserved and they localized around the same regions despite variation in section orientation. Thus, this technique can potentially be applied to study the distribution of ICN neurons in 3D reconstructed hearts of large animals and humans as well as to examine the sex differences and diseased‐induced remodeling of the ICN for future studies.Support or Funding InformationThis work is supported by NIH SPARC Program, grant #3OT2OD023848‐01 (PD/PI: K. Shivkumar; Subaward PIs: J. S. Schwaber, R. Vadigepalli, and Z. J. Cheng)

Direct measurement method of micro-beam X-ray focus based on array detector

A method for directly measuring the focal spot size and minimum focus position output by micro-beam X-ray is proposed. An experimental device based on portable micro X-ray source and capillary X-ray lens is built, and the experiments are carried out at different distances and under different tube voltages. The measured results are compared with the knife-edge scanning method. The results show: the focal spot size and minimum focus position of X-ray lens are 87μm and 9.798 cm respectively by using an array detector. X-ray array detector can directly measure the size of focal spot and predict the position of the minimum focus. It has the advantages of real-time measurement, available two-dimensional distribution, high cost performance and high efficiency. The device can predict the minimum focus position better, but the measured focal spot size is larger than it measured by knife-edge scanning method due to the influence of the pixel size and energy response of the array detector. The measurement results are related to the size of the pixel unit, measurement threshold and exposure time of an array detector.

A Queryable Graph Representation of Vascular Connectivity in the Whole Mouse Brain.

We construct a graph representation for the topology and geometry of the vasculature presenting across the whole mouse brain (dataset: Knife-Edge Scanning Microscope Brain Atlas India Ink). We use our graph representation to calculate preliminary estimates of the average radius as 4:8 μm, total vascular volume as 1:1000 mm3, total vascular surface area as 6:5511 cm2, and total vascular length of 2866:6567 cm. We then isolate a posterior cerebral region, derive its graph representation, and then import that representation to a Neo4j graph database. We then detail how researchers can query this database online to isolate specific vascular networks for further analysis and reconstruction.

Murine Mouse Models of Metastatic Colorectal Cancer in Lungs: Foci Counts in Three Dimensions

In standard histological slide preparations, only a few representative samples are taken, which introduces sampling bias and necessarily eschews information about the full tissue sample. However, it is possible to obtain a full 3D data set of an entire tissue sample using the Knife Edge Scanning Microscope (KESM), an automated simultaneous sectioning and imaging microscope. In this study, we performed manual counts of metastases of colorectal cancer in mouse model lungs stained with India ink and hematoxylin and eosin, which were then sectioned and imaged in their entirety. We were able to get more accurate count of tumor foci than the current standard of surface nodule count can allow; multiple tumor foci were buried hundreds of microns deep into the parenchyma and hence not grossly visible from the surface. Volumetric analysis of whole samples also revealed unusual tumor morphology and permitted the visualization of unique architectural features where multiple tumor foci had grown together. Following the manual count of tumor foci, we developed a semi‐automated algorithm that could increase the efficiency of screening for foci, and create the opportunity for digital 3D reconstruction and morphology interrogation. Without access to a full dataset of the lungs, it is impossible to determine true foci number or morphology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement of the inner diameter of monocapillary with confocal x-ray scattering technology based on capillary x-ray optics.

The inner diameter is one of most important parameters in the design and fabrication of monocapillary x-ray optics, which are widely used in x-ray technology. The confocal x-ray scattering method based on x-ray capillary optics was proposed to nondestructively measure the inner and outer diameters of the monocapillary. The positions of the boundaries of monocapillary x-ray optics were determined by a knife-edge scanning method, and the x-ray optics were profiled in two planes or in three-dimensional space accordingly. This confocal method could conveniently give the inner surface topography of the monocapillary optics, which can be used to show the difference between the actual inner surface and the theoretical one, and has potential applications in nondestructive measurements of stratified substances, or the profile morphology of a specific interface.

Optimized alignment of X-ray mirrors with an automated speckle-based metrology tool.

X-ray mirrors are widely used in beamlines and laboratories as focusing or collimating optics. As well as the highly accurate processes used to fabricate them, optimized alignment of X-ray mirrors also plays an important role in achieving an ideal X-ray beam. Currently, knife-edge scans are the most often used method for aligning X-ray mirrors, which can characterize the focal size and tune the alignment iteratively. However, knife-edge scanning provides only one-dimensional information and this method suffers from being time-consuming and requiring a high-resolution piezo translation stage. Here we describe a straightforward and non-iterative method for mirror alignment by measuring the relationship between the tilt aberration and the misaligned pitch angle, which is retrieved by an at-wavelength metrology technique using a randomly shaped wavefront modulator. Software and a graphical user interface have been developed to automate the alignment process. Combining the user-friendly interface and the flexibility of the at-wavelength metrology technique, we believe the proposed method and software can benefit researchers working at synchrotron facilities and on laboratory sources.

Auto-alignment of X-ray focusing mirrors with speckle-based at-wavelength metrology.

Significant improvements have been made in the fabrication of diffraction-limited X-ray optics used to pursue an aberration-free wavefront. Alignment of these optics plays a crucial role in the resultant beam quality. Here, we present a simple and fast alignment method based on imaging X-ray near-field speckle patterns, with experimental demonstration using a pair of Kirkpatrick-Baez mirrors. The proposed technique has the potential to be an alternative to conventional methods. It loosens the stringent demand for high-resolution scanning stages compared to conventional knife-edge scan and, hence, can be applied to nano-focusing optics. The flexibility and straightforward implementation of the method allow it to be applied to a wide range of experiments at synchrotron facilities and laboratory-based sources.

Tracing Tubular Structures from Teravoxel-Sized Microscope Images.

Tracing vasculature and neurites from teravoxel sized light-microscopy data-sets is a challenge impeding the availability of processed data to the research community. This is because (1) Holding terabytes of data during run-time is not easy for a regular PC. (2) Processing all the data at once would be slow and inefficient. In this paper, we propose a way to mitigate this challenge by Divide Conquer and Combine (DCC) method. We first split the volume into many smaller and manageable sub-volumes before tracing. These sub-volumes can then be traced individually in parallel (or otherwise). We propose an algorithm to stitch together the traced data from these sub-volumes. This algorithm is robust and handles challenging scenarios like (1) sub-optimal tracing at edges (2) densely packed structures and (3) different depths of trace termination. We validate our results using whole mouse brain vasculature data-set obtained from the Knife-Edge Scanning Microscopy (KESM) based automated tissue scanner.

Tracing and analysis of the whole mouse brain vasculature with systematic cleaning to remove and consolidate erroneous images.

Whole mouse brain microvascular images at submicrometer scale can be obtained by Knife-Edge Scanning Microscopy (KESM). However, due to the large size of the image dataset and the noise from the serial sectioning process of the KESM, whole mouse brain vascular reconstruction and analysis with submicrometer resolution have not been achieved yet, while several previous studies demonstrated manually selected small noise-free portion of the KESM dataset. In addition to the KESM dataset, there have been studies for vessel reconstruction and analysis of the whole mouse brain at lower resolution or of partial brain regions at submicrometer resolution. However, to the best of our knowledge, there has been no study for vessel reconstruction and analysis of the whole mouse brain at submicrometer resolution. In this paper, we propose a framework for the 3D reconstruction and analysis of the whole KESM mouse brain vasculature dataset with rich vasculature information extracted at submicrometer resolution. The framework consists of two methods. The propose methods provide the systematic cleaning to remove and consolidate erroneous images automatically, which enables the full tracing and analysis of the whole KESM mouse brain dataset with richer vasculature information.

Robust Cell Detection for Large-Scale 3D Microscopy Using GPU-Accelerated Iterative Voting.

High-throughput imaging techniques, such as Knife-Edge Scanning Microscopy (KESM),are capable of acquiring three-dimensional whole-organ images at sub-micrometer resolution. These images are challenging to segment since they can exceed several terabytes (TB) in size, requiring extremely fast and fully automated algorithms. Staining techniques are limited to contrast agents that can be applied to large samples and imaged in a single pass. This requires maximizing the number of structures labeled in a single channel, resulting in images that are densely packed with spatial features. In this paper, we propose a three-dimensional approach for locating cells based on iterative voting. Due to the computational complexity of this algorithm, a highly efficient GPU implementation is required to make it practical on large data sets. The proposed algorithm has a limited number of input parameters and is highly parallel.

Radiating pattern of surge-current-induced THz light in near-field and far-field zone

We generate the THz wave on the surface of an unbiased GaAs crystal by illuminating femtosecond laser pulses with a 45° incidence angle, and investigate its propagation properties comprehensively both in a near-field and in a far-field zone by performing a knife-edge scan measurement. In the near-field zone, i.e. 540 μm away from the generation point, we found that the beam simply takes a Gaussian shape of which width follows well a behavior predicted by a paraxial wave equation. In the far-field zone, on the other hand, it takes a highly anisotropic shape; whereas the beam profile maintains a Gaussian shape along the normal to the plane of incidence, it takes satellite peak structures along the direction in parallel to the plane of incidence. From the comparison with simulation results obtained by using a dipole radiation model, we demonstrated that this irregular beam pattern is attributed to the combined effect of the position-dependent phase retardation of the THz waves and the diffraction-limited size of the initial beam which lead to the interference of the waves in the far-field zone. Also, we found that this consideration accounting for a crossover of THz beam profile to the anisotropic non-Gaussian beam in the far-field zone can be applied for a comprehensive understanding of several other THz beam profiles obtained previously in different configurations.

Knife-Edge Scanning Microscopy for Bright-Field Multi-Cubic Centimeter Analysis of Microvasculature

Modern light microscopy has benefited from the use of serial section microscopy such as in the Allen Brain Atlas and the creation of new rodent models for Alzheimer’s disease research [1, 2]. Typically, slides are prepared in the conventional manner, where images are collected using traditional light microscopy and digitally scanned. The resulting images then must be computationally registered to one another to create a 3D image. However, numerous issues have inhibited widespread adoption of this method. These include the manual nature of slide preparation and staining, the introduction of artifacts from manual sectioning and mounting on slides, the time taken for whole-slide scanning an entire sample, the difficult nature of image registration and data handling, and the simple fact that it results in too much data for modern infrastructures to handle. The knife-edge scanning microscope (KESM) combines sectioning and imaging into a single step, automates a large part of the traditional pathology workflow, and allows for a greater speed, precision, throughput, and scale at which tissues are digitized (Figure 1). The KESM achieves tissue data collection at a resolution of submicron pixels with a maximum sample volume of over 100 cm3, exceeding the depth offered by confocal or 2-photon microscopy. To handle high-resolution data of over a terabyte per cm3, sophisticated data processing software is applied to model 3D tissue reconstructions, provide interactive image views, and apply quantitative analytics. This allows quantitative analysis to be performed on 3D image stacks of whole mouse organs, which is extremely difficult, expensive, and time-consuming with traditional manual techniques.

Mapping the full vascular network in the mouse brain at submicrometer resolution.

Mapping the microvascular networks in the brain can lead to significant scientific and clinical insights. We developed a serial sectioning microscopy technique called the Knife-Edge Scanning Microscopy (KESM) to section and image the entire mouse brain at submicrometer resolution. In our effort to map the entire vascular network in the mouse brain, we perfused the vessels with India ink, and used KESM to image the prepared brain. This results in about 1.5 TB of raw image data which poses a serious challenge in terms of analysis. In this paper, we will present our data set, and computational algorithms we developed to trace and analyze morphological properties of the mouse brain vascular network. Since the data is available across the entire brain in full detail (the smallest capillaries can be observed in our data), it enables the comparison of regional differences in morphological properties. We expect our results to provide rich insights to brain and neuroscience researchers.

Hard X-ray multilayer zone plate with 25-nm outermost zone width

We have improved the performance of a previously reported multilayer zone plate by reducing its outermost zone width, using the same multilayer materials (MoSi2 and Si) and fabrication technique. The focusing performance was evaluated at the BL24XU of SPring-8 using 20-keV X-rays. The line spread function (LSF) in the focal plane was measured using a dark-field knife-edge scan method, and the point spread function was obtained from the LSF through a tomographic reconstruction principle. The spatial resolution was estimated to be 30 nm, which is in relatively good agreement with the calculated diffraction-limited value of 25 nm, while the measured diffraction efficiency of the +1st order was 24%.