Table 4

Workflow used to digitally process all shark specimens using Avizo (standard 9.2.0)

StepModule/operationSubparameters/inputsJustifications
1 Import the datasets (i.e., each .txm file) for each stain time of a specimen in the project vView• Edit: click preferences• Units tab: tick use and choose correct unitsTo ensure each dataset’s voxel size and unit measure are correct
2 Save project as• File: save as project• Type: Avizo pack & go• Save onto local diskTo save the project now the datasets to work with are imported
Start with CP1-T0
3 Volume rendering• Toolbar: Orthographic viewTo quickly first check your data and make sure you are working with the orthographic view and not the perspective view
4 Extract subvolume• Right click dataset• Search: Extract subvolumeTo crop the data cube to a smaller size (remove the empty space around the head) for faster computation
5 Global axes• View: tick Global AxesTo visualize three-dimensional axes
6 Transform editor• Left click on the green module created in step 4• In the Properties window toolbar: click on Transform Editor• Manually align head to global axes by pressing Shift while moving with the Interact cursor, based on chosen landmarks using Trackball tool in main window toolbarTo align head to a chosen standard position to use relative to global axes
7 Resample transformed image• Right click the green module from step 4• Search: Resample transformed image• Interpolation: Lanczos• Mode: extended• Keep other defaultsTo re-slice each sample according to the global axis and set alignments
8 Extract subvolume• Right click dataset• Search: Extract subvolumeTo crop the data cube again to a smaller size including only the head as the bounding box is bigger after resampling the transformed image
9 Resample• Filter: Lanczos• Mode: voxel size• Resolution: divided by three for x, y, and z values (26.14 → 78.43 μm)To down sample; in this instance, to a third of the initial resolution, to fasten the computations to come
10 Edit new label field• Right click dataset created in step 9• Search: Edit new label field• Threshold tool: select all head and assign to a label• Select the label newly assigned• Toolbar: Selection; Fill; All slices• Repeat filling (previous bullet point) for all panels in the Segmentation Editor, XY, YZ and XZTo select the head and fill the inner volume
Continue with CP1-T1
11 Resample• Reference: T0.resampled• Filter: Lanczos• Mode: voxel sizeTo down sample using the resolution of T0 in step 3, so the computation in the Segmentation Editor is faster
12 Edit new label field• Threshold tool: select all head and assign to a label• Select the label newly assigned• Toolbar: select X; Fill selection; select slice (not 3d volume); fill all slices in the three directions x, y, and z in the four panes windowTo select the head; same as step 10 but in sample at T1
13 Register images• Right click green module CP1-T1.txm (dataset)• Search: Register images• In Properties window:• Model: CP1-T1.labels• Reference: CP1-T0.labels• Transformation: Rigid• Metric: label difference• Keep other defaults• Prealign: click Align lefts, then Align principal axes• ApplyTo register T1-labeled head with T0-labeled head
14 Copy transformation• Data: CP1-T1.txm• Reference: CP1-T1.labelsTo use the previous registration pathway to register the actual dataset
15 Resample transformed image• Right click green module CP1-T1.txm (dataset)• Search: Resample transformed image• Data: CP1-T1.txm• Interpolation: Lanczos• Mode: cropped• ApplyTo re-slice according the alignment of reference T0
REPEAT steps 11–15 for each other staining time Ti .
Then, do the following for all staining times
16 ROI box• Right click on each green module created in step 10• Search: ROI box• Create for each sample• Shift select each of the ROI box• Click on the Connection Editor on all of them in the Properties window• Click and drag to link all the Minimum [unit] and Maximum [unit] from the reference ROI Box to the nextTo select the same area including only the head for all samples
17 Crop editor• Left click on the green module created in step 10• In the Properties window toolbar: click on Crop Editor• In the small window that opens; Crop list: tick “Use ROI list”• Resolution mode: bounding boxTo crop all ROI boxes to the same volume
18 Orthoslice• Right click on each green module created in step 15• Search: Orthoslice to create oneTo check all orthoslices are matching in all three views (XY, XZ, and YZ) in the main display window
19 Resample• Data: green module obtained in step 15• Reference: NO SOURCE• Filter: Lanczos• Mode: voxel size• Voxel size [units]: doubledTo down sample by half and speed up filtering computations in the next step
20 Non-local means filter• Right click on each green module created in step 15• Search: Non-local means filter• Keep all defaults but make Filter: XY planes is selected• ApplyTo smooth or reduce noise
21 Orthoslice• Right click on each green module created in step 20• Create four orthoslices for each• Set Colormap to standardized range for allTo position the four sampling slices in transversal view at the position of interest in the brain
22 Line probe• Right click on each green module created in step 20• Search: Line probe and create four probes• Enter Slice number corresponding to slices chosen in step 21• Sampling options: number of samples 500To position the four lines probes corresponding to each orthoslice using the same coordinates as the reference line probe chosen for all times
Select each orthoslice and corresponding line probe sequentially to download the data.