| Mouse.number | Element | Molecular.Weight | Injection.Site | Contrast.agent.commercial.name |
|---|---|---|---|---|
| 21 | Iodine | High MW | ventricle | ExiTron P |
| 23 | Gadolinium | High MW | ventricle | GadoSpin P |
| 24 | Gold | Nanoparticles | ventricle | Aurovist 15 nm |
| 22 | Gadolinium | Low MW | ventricle | Gadodiamide |
| 19 | Barium | Nanoparticles | ventricle | ExiTron nano 12000 |
| 30 | Gold | Nanoparticles | cisterna magna | Aurovist 15 nm |
| 25 | Barium | Nanoparticles | cisterna magna | ExiTron nano 12000 |
In vivo Imaging of Central Nervous System Fluid Spaces using Synchrotron Radiation Micro-Computed Tomography
Marta Girona Alarcón (first author) 
Vartan Kurtcuoglu (last author)
In this hub, we provide additional materials, methods and results for the publication.
Downloads
In this DOI, we provide full resolution datasets for download from the publication data, as well as complete metadata and additional materials to reproduce the experiments.
Supplemental Results
Pilot post mortem experiments
In a previous beamtime (ESRF, 2020, December), ex vivo imaging was performed. The goal of the pilot experiments was to chose a contrast agent for the presented in vivo experiments. The list of used contrast agents is available under the Zenodo download entry “tables_exVivo.7z”. We considered the Barium-based contrast agent (CA) to be the most promissing one and therefore decided to employ it for the in vivo experiments (Fig. 1).
Fig. 1. Ex vivo coronal slices of three different mice injected with (left) Mouse.number 23, Gadolinium-based, (middle) Mouse.number 19, Barium-based and (right) Mouse.number 30, Gold-based contrast agent.
Table 1. List of ex vivo mouse number, including the injected site and contrast agent.
CSF spaces segmentation
In the publication, we have shown the need of infusing contrast agent to achieve a semi-automatic segmentation. In the figure below we show a rendered segmentation. Infusion of contrast agent in the lateral ventricles during high-resolution synchrotron radiation-based hard X-ray computed tomography imaging allows 3D visualization with virtual slicing in all directions. In the downloads, we have provided a segmentation of a timeseries imaging in the brain ventricles, which can be found under “videos_Reconstructions.7z”.
Fig. 2. Three dimensional rendering of CSF spaces enclosed in the mouse skull. Lateral ventricles in blue, third ventricle in green, aqueduct in pink and fourth ventricle in yellow.
Calibration ramp
To quantify spatial CA distribution, we first determined the relation between image intensity and barium concentration, finding the thepretically expected linear relation. The single points to obtain the linear relation were obtained from 3D segmentation of the tubes available in Fig. 3.
Fig. 3. In vitro calibration scans used for the calibration ramp presented in Experiment 3. Tubes containing different dilutions of CA resulting in a final concentration of 0, 48, 120, 158.4 and 240 mg Ba/mL (left to right).
Timeseries movies
Movement of contrast agent over time for the presented timeseries can be observed in projections and reconstructions as movies under the folders “videos_Reconstructions.7z” and “videos_Projections.7z” in Zenodo.
Additional materials
We have additively manufactured the mouse holder that is optimized for vertical imaging while ensuring fixation of the mouse skull (Fig. 4). As it can be seen in the right side, the body part of the holder has water channels that facilitate a good physiological temperature for the animal.
Fig. 4. (Left) Visualization of the mouse holder mounted on the imaging rotational stage. (Right) Translucent visualization of body mouse holder part containing the water channels to ensure physiological temperature of the mouse.
For the surgery room, we assambled a customized animal box to increase the starting body mouse temperature. We set the desired temperature to 33°C. Additionally, to facilitate the surgical interventions, we manufactured a mouse stage for tracheotomy and cisterna magna infusion (Fig. 5).
Fig. 5. (Left) Customized prewarm box consisting of an anesthesia induction chamber attached to two reptile generic warming pads, which are connected to an automatic temperature controller. (Right) Customized tracheotomy and cisterna magna stage. Available as a 3D model in the downloads. ## Equipment and consumables
The following table provides details of consumables, hardware and software that have been employed for this experiments.
Links to protocol pages
All protocols can be found this website’s repository: https://gitlab.uzh.ch/snf_213535/fdcns
Sample information
/builds/gorka.fragagonzalez/fdcns_fork/webpage_contents/publications/20240901_Marta_GironaAlarcon
Codebook
| Variable | Description |
|---|---|
| Subject_ID | Unique identifier for each animal |
| Date | Date of the experiment. Format YYYY-MM-DD |
| Preparation | Indicator whether the experiment was in vivo, ex vivo, post mortem or peri mortem. This reflects the state of the animal at the beginning of the scan (e.g. animals that are scanned first in vivo but are scanned again post mortem are indicated as in vivo. Those post mortem scans will be indicated as such on the Scan_List) |
| Contrast_agent_Injection_location | Anatomical region where X-ray contrast agent was injected for the experiment |
| Comments | Text comments indicating additional information |
| Sex | Sex of the animal |
| Body_weight | Body weight of the animal on the date of the experiment |
| Date_of_birth | Date of birth of the animal. Format YYYY-MM-DD |
| Strain | Strain from which the mouse line is derived (e.g. C57BL/6J; C57BL/6JJmsSlc; B6D2F1/J) |
| Genetic_strain_type | Strain type of transgenic animals. Indicated as wild-type if non-transgenic |
| Pathology | Description of pathology or treatment group if experiment investigates disease model |
| Stereotactic_coordinates_shorthand | Shorthand for a set of stereotactic coordinates used for intraventricular contrast agent injection. Refers to the place coordinates were first used by the FABRIC4 consortium (e.g. TKI; Spring-8; CLS) |
| Stereotactic_coordinate_X | Stereotactic coordinate on the lateral-medial axis. Indicates offset of the injection location from the bregma. Negative values point to the right and positive values point to the left direction of the animal. In the context of injecting the right ventricle: right = lateral; left = medial |
| Stereotactic_coordinate_Y | Stereotactic coordinate on the posterior-anterior axis (or caudal-rostral axis). Indicates offset of the injection location from the bregma. Negative values point to the posterior (caudal) and positive values point to the anterior (rostral) direction of the animal |
| Stereotactic_coordinate_Z | Stereotactic coordinate on the ventral-dorsal axis. Indicates depth of the injection from the skull surface as determined by stereotact. Negative values point to the ventral and positive values point to the dorsal direction of the animal |
| Infusion_cannula_length | Length of the needle part of plastic infusion cannulas used for contrast agent infusions. This corresponds to the depth of the infusion site with respect to the skull surface |
| Contrast_agent_concentration | Concentration of the main X-ray absorbing element in the contrast agent (e.g. mg I/ml; mg Ba/ml) |
| Contrast_agent_infusion_rate | Amount of contrast agent volume per time unit infused for the main experiment |
| Contrast_agent_maintenance_infusion_rate | Amount of contrast agent volume per time unit infused after the main infusion. Low infusion rates are maintained to avoid backflow into the contrast agent cannula. |
| Contrast_agent_infusion_volume | Overall amount of contrast agent volume infused for the main experiment. Volume of maintenance infusion are not included in that number |
| Contrast_agent_infusion_start_time | Time of day when contrast agent infusion was started. Format HH:MM |
| Contrast_agent_infusion_started_before_scan_name | Filename of first scan after contrast agent infusion was started |
| Anesthesia_induction | Anesthetic regimen used for anesthesia induction |
| Anesthesia_maintenance | Anesthetic regimen used for anesthesia maintenance during surgery or imaging |
| Analgesia | Analgesia regimen used |
| Artificial_ventilation | Artificial ventilation used (e.g. tracheotomy; intubation) |
| Research_group_institution | University affiliation of the group(s) who collected the data. (Unibas = University of Basel; Unibe = University of Bern; UZH = University of Zurich) |
| Research_group | Abbreviation describing the group(s) who collected the data. (BMC = Biomaterials Science Center; TKI = Theodor Kocher Institute; TIG = The Interface Group) |
| Table_date_csv_export | Date of exporting the .xlsx table used for editing to .csv for upload to the FABRIC4 Portal. |
| N/A | Not applicable |
| Unavailable | This information has not been collected |
| Preview_Image | Preview thumbnail of the dataset provided on the FABRIC4 Portal for navigating data |
| Preview_Image_Slice_Number | Preview thumbnail is typically a single slice of a 3D image stack. Slice number indicates the location of the 2D image slice in the 3D stack. |
| Date | Date of the data acquisition. Format YYYY-MM-DD |
| Tissue_ID | Unique identifier for each sample. A single animal may provide multiple tissue samples |
| Anatomical_location | Anatomical location of the scan or of the tissue source |
| Filename | Name of file or folder containing the data |
| Filename_changes | Filename may occasionally be incorrectly assigned during the experiment. Changes made to the original filename are indicated here. |
| Number_of_subscans_in_file | Number of scans included in the file or folder indicated in the Filename column |
| Preparation | Indicator whether the experiment was in vivo, ex vivo, post mortem or peri mortem |
| Tomography_acquisition_scheme | Description of acquisition scheme used impacting the field of view (e.g. half-acquisition; mosaic tomography) |
| Tomography_scan_type | Description of the type of scan impacting reconstruction (e.g. height steps; time series; gated scan) |
| Repository | Name of the public repository where the data indicated in Filename can be downloaded |
| Repository_URL | URL linking to the entry in the public repository where the data indicated in Filename can be downloaded |
| Mean_photon_energy | Mean photon energy of the X-ray beam |
| Monochromator | Type of monochromator used for narrowing the energy spectrum of the X-ray beam |
| Effective_voxel_size | Effective voxel size of the data. This value derives from the lens magnification and Detector_pixel_size. It is usually measured independently for validation |
| Projections_width_in_pixel | Width of the acquired X-ray projection in pixels |
| Projections_height_in_pixel | Height of the acquired X-ray projection in pixels |
| Projections_width | Width of the acquired X-ray projection in millimeters |
| Projections_height | Height of the acquired X-ray projection in millimeters |
| Exposure_time | Amount of time that the camera chip used in the X-ray detector setup is exposed to light and collecting photons |
| Overhead_time_per_projection | Additional amount of time required on top of the exposure time for the complete acquisition of an X-ray projection (e.g. for readout or rotation stage movements) |
| Number_of_projections | The number of X-ray projections collected at multiple angles for the purposes of tomographic reconstruction |
| Stage_rotation_angle_range | Indicates the angle the rotation stage was rotated during the full scans. Typical values are 180 ° and 360 ° |
| Stage_position_z | Absolute motor position of the motor used for translating the height of the sample. This value is useful if height steps are not acquired sequentially |
| Height_step_size | Absolute motor position of the motor used for translating the height of the sample between different height steps |
| Propagation_distance | Distance between the detector and the sample. This value has impacts the amount of edge enhancement seen in the X-ray images |
| Detector_model | Name identifying the camera used in the detector setup |
| Detector_pixel_size | Pixel size of the physical pixels on the camera chip without magnification |
| Detector_width_in_pixel | Width of the camera chip array in number of pixels. This reflects the technical specifications of the camera chip and not the number of pixels used in imaging |
| Detector_height_in_pixel | Height of the camera chip array in number of pixels. This reflects the technical specifications of the camera chip and not the number of pixels used in imaging |
| Lens_type | Type of lens used for magnification of visible light in the X-ray detector setup |
| Lens_f-number | The f-number of a camera lens used for magnification in the X-ray detector setup. Indicated as N/A for microscope objectives where the numerical aperture is provided instead |
| Lens_numerical_aperture | The numerical aperture of a microscope objective used for magnification in the X-ray detector setup. Indicated as N/A for camera lenses where the f-number is provided instead |
| Scintillator | Material and thickness of the scintillator used to convert X-rays to visible light in the X-ray detector setup |
| Radiation_dose_rate | Radiation dose rate measured or estimated for in vivo experiments. This metric is not measured for ex vivo or post mortem experiments |
| Facility_name | Name or acronym of the facility used for data acquisition (e.g. core facility; synchrotron) |
| Facility_subunit | Name or acronym of the subunit of the facility used for data acquisition (e.g. beamline of a synchrotron) |
| Facility_institution | Name or acronym of any overarching organization hosting the facility used for data acquisition (e.g. university hosting core facility; radiation research organization) |
| Facility_country | Country where the facility is located |
| Beamtime_start_date | Date of the first official day of the synchrotron beamtime allocation. This does not include any days prior of official beamtime start used for preparation. Format YYYY-MM-DD |
| Beamtime_proposal_number | Official proposal number assigned by the synchrotron to the allocated beamtime. This should correspond the number used in the synchrotron databases etc. |
| Beamtime_project_description | Shorthand description of the research question investigated during this experiment |
| Disk_ID_Projections | Unique identifier for the physical harddisk used to store the raw X-ray projection data of the scan indicated in Filename. Format Synchrotron_MonthYear_Disk# |
| Disk_ID_Reconstructions | Unique identifier for the physical harddisk used to store the reconstructed 3D data of the scan indicated in Filename. Format Synchrotron_MonthYear_Disk# |
| Disk_Basel_Physical_Location | Description of the physical location of the harddisks stored at the Biomaterials Science Center at the University of Basel |
| Disk_Bern_Physical_Location | Description of the physical location of the harddisks stored at the Theodor Kocher Institute at the University of Bern |
| Disk_Zurich_Physical_Location | Description of the physical location of the harddisks stored at The Interface Group at the University of Zurich |
Codebook
| Variable | Description |
|---|---|
| Subject_ID | Unique identifier for each animal |
| Date | Date of the experiment. Format YYYY-MM-DD |
| Preparation | Indicator whether the experiment was in vivo, ex vivo, post mortem or peri mortem. This reflects the state of the animal at the beginning of the scan (e.g. animals that are scanned first in vivo but are scanned again post mortem are indicated as in vivo. Those post mortem scans will be indicated as such on the Scan_List) |
| Contrast_agent_Injection_location | Anatomical region where X-ray contrast agent was injected for the experiment |
| Comments | Text comments indicating additional information |
| Sex | Sex of the animal |
| Body_weight | Body weight of the animal on the date of the experiment |
| Date_of_birth | Date of birth of the animal. Format YYYY-MM-DD |
| Strain | Strain from which the mouse line is derived (e.g. C57BL/6J; C57BL/6JJmsSlc; B6D2F1/J) |
| Genetic_strain_type | Strain type of transgenic animals. Indicated as wild-type if non-transgenic |
| Pathology | Description of pathology or treatment group if experiment investigates disease model |
| Stereotactic_coordinates_shorthand | Shorthand for a set of stereotactic coordinates used for intraventricular contrast agent injection. Refers to the place coordinates were first used by the FABRIC4 consortium (e.g. TKI; Spring-8; CLS) |
| Stereotactic_coordinate_X | Stereotactic coordinate on the lateral-medial axis. Indicates offset of the injection location from the bregma. Negative values point to the right and positive values point to the left direction of the animal. In the context of injecting the right ventricle: right = lateral; left = medial |
| Stereotactic_coordinate_Y | Stereotactic coordinate on the posterior-anterior axis (or caudal-rostral axis). Indicates offset of the injection location from the bregma. Negative values point to the posterior (caudal) and positive values point to the anterior (rostral) direction of the animal |
| Stereotactic_coordinate_Z | Stereotactic coordinate on the ventral-dorsal axis. Indicates depth of the injection from the skull surface as determined by stereotact. Negative values point to the ventral and positive values point to the dorsal direction of the animal |
| Infusion_cannula_length | Length of the needle part of plastic infusion cannulas used for contrast agent infusions. This corresponds to the depth of the infusion site with respect to the skull surface |
| Contrast_agent_concentration | Concentration of the main X-ray absorbing element in the contrast agent (e.g. mg I/ml; mg Ba/ml) |
| Contrast_agent_infusion_rate | Amount of contrast agent volume per time unit infused for the main experiment |
| Contrast_agent_maintenance_infusion_rate | Amount of contrast agent volume per time unit infused after the main infusion. Low infusion rates are maintained to avoid backflow into the contrast agent cannula. |
| Contrast_agent_infusion_volume | Overall amount of contrast agent volume infused for the main experiment. Volume of maintenance infusion are not included in that number |
| Contrast_agent_infusion_start_time | Time of day when contrast agent infusion was started. Format HH:MM |
| Contrast_agent_infusion_started_before_scan_name | Filename of first scan after contrast agent infusion was started |
| Anesthesia_induction | Anesthetic regimen used for anesthesia induction |
| Anesthesia_maintenance | Anesthetic regimen used for anesthesia maintenance during surgery or imaging |
| Analgesia | Analgesia regimen used |
| Artificial_ventilation | Artificial ventilation used (e.g. tracheotomy; intubation) |
| Research_group_institution | University affiliation of the group(s) who collected the data. (Unibas = University of Basel; Unibe = University of Bern; UZH = University of Zurich) |
| Research_group | Abbreviation describing the group(s) who collected the data. (BMC = Biomaterials Science Center; TKI = Theodor Kocher Institute; TIG = The Interface Group) |
| Table_date_csv_export | Date of exporting the .xlsx table used for editing to .csv for upload to the FABRIC4 Portal. |
| N/A | Not applicable |
| Unavailable | This information has not been collected |