A wide-field micro-computed tomography detector: micron resolution at half-centimetre scale

Abstract

Ideal three-dimensional imaging of complex samples made up of micron-scale structures extending over mm to cm, such as biological tissues, requires both wide field of view and high resolution. For existing optics and detectors used for micro-CT (computed tomography) imaging, sub-micron pixel resolution can only be achieved for fields of view of <2 mm. This article presents a unique detector system with a 6 mm field-of-view image circle and 0.5 µm pixel size that can be used in micro-CT units utilizing both synchrotron and commercial X-ray sources. A resolution-test pattern with linear microstructures and whole adult Daphnia magna were imaged at beamline 8.3.2 of the Berkeley Advanced Light Source. Volumes of 10000 × 10000 × 7096 isotropic 0.5 µm voxels were reconstructed over a 5.0 mm × 3.5 mm field of view. Measurements in the projection domain confirmed a 0.90 µm measured spatial resolution that is largely Nyquist-limited. This unprecedented combination of field of view and resolution dramatically reduces the need for sectional scans and computational stitching for large samples, ultimately offering the means to elucidate changes in tissue and cellular morphology in the context of larger, whole, intact model organisms and specimens. This system is also anticipated to benefit micro-CT imaging in materials science, microelectronics, agricultural science and biomedical engineering.

Keywords: X-ray optics; high resolution; histotomography; large field of view; synchrotron micro-CT.

Figure 1

Generalized beam alignment and image acquisition setup for application in synchrotron facilities and tabletop sources. CAD design (a). Actual setup as assembled in the Berkeley ALS 8.3.2 beamline hutch (b). Our custom detector operates using a scintillator (A¹) to convert X-rays (A²) passing through a sample (A³) into visible light. The fluorescence image is then focused by the objective lens (A⁴) onto the CMOS sensor surface (A⁵). Commercially available Newport stages (A⁶–A⁸) were utilized to adjust the camera relative to the focal point, and to translate the detector through three-dimensional space (15 cm vertical travel and 10 cm horizontal travel in any direction) for optimal alignment with any synchrotron- or tabletop-generated beam. Projections (A⁹) acquired by the detector are saved using the Harvester Python library and later reconstructed with the TomoPy toolbox in Python.

Figure 2

Field-of-view characterization utilizing the ALS synchrotron beam and large-scale biological sample. The entirety of the beam height at the ALS 8.3.2 beamline is encapsulated by the detector’s field of view while maintaining 0.5 µm pixel size (a). A 2000 × 2000 pixel field of view at the same 0.5 µm pixel size is shown in red. To illustrate the field of view in a more biologically applicable and practical sense, we present a cm-scale, juvenile, 57 day post-fertilization (d.p.f.) zebrafish embedded in resin and stained with phospho­tungstic acid (PTA) (b) using a local NOVA 96000 tungsten filament source to demonstrate field of view (c), with coverage of the same 2000 × 2000 pixel array in red and our detector’s field of view in blue. Rotating the camera by 90° makes it possible to scan in a single view horizontally and to stitch in four sections vertically, compared with approximately 500 scans on a 2000 × 2000 detector to cover the same volume. Red dotted lines indicate areas of field-of-view overlap.

Figure 3

Reconstruction of QRM nanobar phantom die acquired over a single projection series. Projection from the side including both case dies (a). The zoomed-in elements of the reconstructed vertical die (b′) qualitatively demonstrate the resolution of elements of defined size. Patterns including line pairs and dots ranging from 10 µm to 1 µm (c′) are shown, along with a dot pattern containing elements that are 2 µm in diameter (e′), an etched QRM symbol (f′), and the reconstruction of the same Siemens star (d′) previously shown as a projection in Fig. S1.

Figure 4

Verification of resolution utilizing a QRM bar pattern phantom. A QRM-manufactured phantom including line and point patterns, Siemens stars and an L edge (a) was scanned at the Berkeley ALS and locally reconstructed to characterize the resolution of the combined lens system and camera detector. A portion of the reconstructed phantom is presented (b) showcasing line pairs ranging from 10 to 1 µm in width. A line profile of the indicated regions is shown, validating resolution up to 1 µm (c). A smaller line profile taken across the 1 µm line pairs that highlights signal shape is shown (d). Signal at the edges of line pairs is enhanced by phase effects caused by the silicon–air interface. Pixel counts between line pairs confirm reconstruction at 0.5 µm, as each line of the 1 µm pattern is 2 pixels thick.

Figure 5

Slanted edge response and calculation of MTF in the projection domain. A 400 projection series taken at the same angle of the indicated area of the slanted L edge (a) was used to record the associated averaged intensity line profile (b). The LSF (c) was taken as the first derivative of (b) across pixel distance, and the MTF (d) was calculated as the discrete normalized Fourier transform of the portion of (c) covering the edge. The full width at half-maximum amplitude of the LSF was recorded at 0.90 µm (c), with the indicated 0.1 MTF amplitude located at 0.87 line pairs per µm (d). Line pairs are presented with a corresponding averaged intensity line profile indicating contrast within the 10, 5, 4, 3, 2 and 1 µm line pair groups (e). Edge enhancement can be visualized most easily in the intensity profile region corresponding to the 10 µm demarcations.

Figure 6

Adult D. magna stained with PTA showing sub-cellular resolution for different cell types and structures (a). Cell types and structures that can be visualized by customizing various stack thicknesses included: muscle striations (about 2 µm) on the labral muscles (b), ommatidia (Om), optic nerves (ON), optic lobe (OL) and brain (Br) (c), setae of the filter plates (d), nucleoli (yellow arrows, ∼2 µm diameter) in the gut epithelial cells (red arrows) (e), nucleoli (green arrows) in the fat cells (f) and yolk globules (g) in a developing embryo. Panels (a) to (c) were generated from maximum intensity projections (MIP) of 50 slices, panel (d) from an MIP of 100 slices to visualize thicker three-dimensional structures and panels (e) to (g) from an MIP of ten slices. An MIP of ten slices is equivalent to a 5 µm-thick histology slide.