Synchrotron radiation-based micro computed tomography dataset - in vivo mouse brain - Mouse50

Intra-cisterna magna infusion at ESRF ID17 beamline

This datasets features a synchrotron radiation-based micro computed tomography dataset of a C57BL/6J mouse injected with barium-based contrast agent into the cisterna magna. Data was recorded at the beamline ID17 of the European Synchrotron Radiation Facility in June 2022. For more information, see the associated publication, repository or the FABRIC4 portal.

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_001.zipFull 3D-stack, time point 1, start of contrast agent infusion

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_012.zipFull 3D-stack, time point 12, 5.5 min after contrast agent infusion start, 0.5 min after end of infusion

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_slice0350_timesteps01-502D slice z = 350, all time points

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_slice0350_timesteps01-50_registered2D slice z = 350, all time points, registered and transformed tomograms

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_slice0500_timesteps01-502D slice z = 500, all time points

mouse50_male_alive_23p7g_0p1ulmin_2p5ul_2KTimeResolved_cisternaMagna_timeseries2_slice0500_timesteps01-50_registered2D slice z = 500, all time points, registered and transformed tomograms

Supplementary_Video_4.mp42D slice z = 500, all time points, registered and transformed tomographs

Methods

For the dataset shown, a male mouse (SubjectID: Mouse50) twelve weeks of age and 23.7 g body weight was first injected subcutaneously with buprenorphine (0.1 mg/kg) for analgesia. Anesthesia was induced after onset of analgesia 30 min later, via intraperitoneal injection of a cocktail of ketamine (73 mg/kg) and medetomidine (0.18 mg/kg). The depth of anesthesia was monitored by testing reflexes, and additional injections were given as needed. During imaging, this was done via an intraperitoneal infusion line (30G needle with 0.28 mm inner diameter tubing) connected to a remote-controlled syringe pump. 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.

The cisternal infusion system consists of a 30G needle connected via tubing to a 1 ml syringe, following the procedure described by Xavier et al., with minor modification. The needle was carefully cut using a small rotary saw, instead of breaking off the needle with a bevel of an insulin syringe. To ensure removal of any metal residues from the sawing process, the needle was rinsed by submerging it in water and then thoroughly flushed. Before the start of infusion implant surgery, syringes and tubing were mounted into the syringe pump and filled with mineral oil as a hydraulic fluid, ensuring that no air bubbles were introduced. To prevent the mixing of fluids, 2 μl of air was drawn between the hydraulic fluid and the contrast agent, preventing a mixed fluid interface. 1.5× concentrated contrast agent (480 mg Ba/ml) was then drawn into the system shortly before implantation, to avoid drying or the introduction of air bubbles.

The mouse was secured in a stereotactic frame and a small skin incision was made over the occipital bone. The three muscle layers covering the cisterna magna were carefully dissected under a stereomicroscope using fine forceps and scissors. The atlantooccipital membrane was then perforated, and the cannula needle was gently inserted into the cisterna magna. A metal clip was used to stabilize the cannula during insertion and was gently removed once the cannula was securely in place and sealed with tissue glue.

The animal was then transferred to the radiation hutch containing the SRµCT imaging setup described above, where it was imaged with a monochromatic beam at a photon energy of 37.5 keV. 2000 radiographs over a rotation range of 360° were acquired with a pco.edge 5.5 camera coupled with a Hasselblad 100 mm f/2.2 lens and a 250 µm LuAg:Ce scintillator for 6.45 µm effective pixel size. The field of view was reduced to 2560 × 700 pixels due to the limited height of the X-ray beam, and recorded with 4 ms exposure time and 1 ms overhead time. Acquisition time per scan was 10 s. Sample-detector distance was 2.5 m. The animal was infused with contrast agent while one tomographic scan was acquired every 30 seconds for 25 min. 2.5 µl of contrast agent were infused at rate of 0.5 µl/min in the first 5 min of the time series, then infusion was stopped. Flat-field images were only acquired at the beginning and the end of the 50 scans.

Tomograms were reconstructed using the ESRF software Nabu (version 2023.2.0), and its filtered backprojection algorithm. Stripe removal in the sinograms was achieved using a combined wavelet-Fourier filtering. The center of rotation for each reconstruction was manually fine-tuned.

In order to isolate potential motion of the ventricles from the effect of whole animal motion, data volumes of the different time points used for comparison were registered to the first reconstruction of the series (called reference image) via rigid registration. An extended bone mask without ventricular spaces was created for the reference image to ensure that the contrast enhancement of the ventricles would not affect registration. The registration was driven by maximizing normalized cross-correlation as an image similarity measure within the extended bone mask. Automatic image registration used the open-source software elastix (version 4.9). Images were rescaled to the intensity range in the ROI of the reference image to reduce quantification errors when using 256 bins for covering the intensity range during registration. Images of the original intensities were afterwards transformed based on the registration result.

Due to an incorrect voxel size of 6.0 µm being entered in the metadata and used during reconstruction, the intensity values in the datasets provided were multiplied by 6.0 µm / 6.45 µm = 0.930 after reconstructions so that the intensity values correspond to linear attenuation coefficients.