DOI: 10.5281/zenodo.15671326
title: Synchrotron radiation-based micro computed tomography dataset - in vivo mouse brain - JP28

description: Contrast-enhanced imaging at SPring-8 BL20B2 beamline

This dataset features a synchrotron radiation-based micro computed tomography dataset of a C57BL/6JJmsSLC mouse injected with barium-based contrast agent into the right lateral ventricle. Data was recorded at the beamline BL20B2 of the synchrotron SPring-8 in May 2023. For more information, see the associated publication, repository or the FABRIC4 portal.



JP28-invivo-5scan-phase3-postinjection-brain_raw_003.zipFull 3D-stack, time point 3, 40 min after start of contrast agent infusion


To correlate contrast agent concentrations with the linear attenuation coefficients, calibration scans were performed using different concentrations of the contrast agent. These data are available in the following repository:



Contrast agent calibration phantomshttps://doi.org/10.5281/zenodo.15675683


Methods

C57BL/6JJmsSLC mice were supplied by Japan SLC, Inc. To ensure proper acclimatization, the animals were housed within the local animal facility and habituated to non-aversive handling techniques 10 days before the experiments. The experimental protocols were reviewed and approved by the responsible ethics committee of SPring-8.

For the dataset shown, a female mouse (SubjectID: JP28) twelve weeks of age and 20.2 g body weight was first anesthesized with a cocktail of medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol (5 mg/kg). In order to prevent hypothermia, the entire surgical area and radiation hutch was heated to a temperature of 32 °C. The depth of anesthesia was monitored by testing reflexes, and additional intraperitoneal injections were given as needed. Eye ointment was applied and the skull, neck, and upper thoracic region of the mouse were shaved to avoid potential artefacts during X-ray imaging. 2 × 0.5 ml glucose 10% was administered in two separate subcutaneous injections, and tracheotomy was performed using a metal tracheal cannula with Y-adapter for artificial ventilation.

The mouse was then secured in a stereotactic frame and ventilated with a MiniVent (Model 845, Hugo Sachs Elektronik, March-Hugstetten, Germany), providing 96% oxygen from an oxygen accumulator device and 0.5 – 2% isoflurane. The exhaled air was routed through a water column to maintain 2 cmH2O positive end-expiratory pressure. Stroke volume was set to 125 µl, respiration rate to 150 bpm. Buprenorphine (0.1 mg/kg) for analgesia was injected subcutaneously after the animal was under isoflurane anesthesia, as buprenorphine is a partial antagonist of butorphanol.

The ventricular infusion system consisted of plastic tubing with an outer diameter of 1.14 mm and an inner diameter of 0.69 mm. One end of the tubing was connected to a 25 µl Hamilton syringe using a 1 mm compression fitting. The other end was glued to an MRI-compatible PEEK infusion 28G microcannula with a custom 2.3 mm cannula length. The infusion was controlled using a syringe pump. Prior to the infusion implant surgery, the syringes were mounted in the syringe pump and, together with the connected tubing, filled with water as a hydraulic fluid. Care was taken to ensure that no air bubbles were present in the system. To prevent the mixing of fluids, 2 μl of air was withdrawn between the hydraulic fluid and the contrast agent, preventing a mixed fluid interface. Contrast agent diluted to 240 mg Ba/ml was then drawn into the system shortly before implantation, to avoid drying or the introduction of air bubbles.

For cannula implantation, excess periosteum from the bone was removed and the bregma was identified. A small hole of about 1 mm diameter was drilled through the parietal bone at the injection coordinates. The microcannula was carefully inserted into the right lateral ventricle (Fig. 4a) using the stereotactic frame, following established methods . Medio-lateral and rostral-caudal coordinates for cannula insertion were adjusted to account for anatomical differences in local mouse strains and variability among different surgeons, and was 1.2 mm lateral and 0.22 mm caudal of the bregma.

The animal was then transferred to the radiation hutch containing the SRµCT imaging setup, where it was imaged at a photon energy of 40 keV, provided by the beamline’s double multilayer monochromator with 4.8% energy bandwidth. 1800 radiographs over a rotation range of 180° were acquired with a Hamamatsu Orca Flash4.0 v2 camera coupled to a tandem lens system (105 mm f/2.4 and 85 mm f/1.4) and a 500 µm LuAG:Ce scintillator for 8.0 µm effective pixel size. The field of view was reduced to 2048 × 1500 pixels (16.4 mm × 12.0 mm) due to the limited height of the X-ray beam, and images were recorded with 5 ms exposure time and 8 ms overhead time. Acquisition time per scan was 23 s. The distance between sample and detector was 1 m.

The animal was infused with contrast agent while mounted in the radiation hutch, to allow simultaneous imaging. Contrast agent flow rate was ramped up linearly from 0 to 0.25 µl/min within the first minute and kept until 5 µl of contrast agent were infused. To avoid backflow of CSF into the infusion cannula, a very low flow rate of 0.02 µl/min was maintained thereafter. The dataset shown in Fig. 2b was acquired 40 min after the start of the infusion, after a total of 5.39 µl of contrast agent were infused.

Tomograms were reconstructed using BL20B2’s in-house reconstruction software ct-rec (version 2023.05.03), using automatic center-of-rotation determination. Output image intensity values directly correspond to linear attenuation coefficients.

To correlate contrast agent concentrations with the linear attenuation coefficients, calibration scans were performed using different concentrations of the contrast agent. These solutions were prepared by diluting a customized 480 mg Ba/ml (1.5× concentrated compared to stock product, 320 mg Ba/ml) suspension with isotonic mannitol solution (54.65 mg/ml, the same suspension medium as used by the manufacturer) to achieve final concentrations of 0, 48, 120, 158.4 and 240 mg Ba/ml. At each concentration, 4 µl of solution were drawn by a syringe pump into a 0.69 mm inner diameter PVC tube. These concentration samples were then imaged with the same image acquisition settings as described above. The volumes containing the solutions were segmented using manual contouring in Python, employing the morphological_geodesic_active_contour algorithm available in the scikit-image package. This process enabled the determination of mean gray values, which correspond to the mean X-ray linear attenuation coefficients.