Workflow used to digitally process all goldfish specimens using Avizo (standard 9.2.0)
| Step | Module/operation | Subparameters/inputs | Justifications |
|---|---|---|---|
| 1 | Import the datasets (i.e., each .txm file) for each stain time of a specimen in the Project View | • Edit: click preferences• Units tab: tick use and choose correct units | To ensure each dataset’s voxel size and unit measure are correct |
| 2 | Save project as | • File: save as project• Type: Avizo pack & go (.am format)• Save onto local disk | To save the project now the datasets to work with are imported |
| Start with GF1-T0 | |||
| 3 | Volume rendering | • Right click green dataset• Search: Volume rendering• Toolbar: Orthographic view | To 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 subvolume | To crop the data cube to a smaller size (remove the empty space around the head) for faster computation |
| 5 | Global axes | • View: tick Global 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 toolbar | To 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 defaults | To re-slice each sample according to the global axis and set alignments |
| 8 | Extract subvolume | • Right click dataset• Search: Extract subvolume | To crop the data cube again to a smaller size including only the head as the bounding box is bigger after resampling the transformed image |
| Continue with GF1-T1 | |||
| 9 | Register images | • Right click green module GF21-T1.txm• Search: Register images• In Properties window:• Model: GF1-T1.labels• Reference: GF1-T0.labels• Keep all defaults below• Transformation: Rigid• Metric: Normalized mutual information• Prealign: click Align lefts, then Align principal axes• Click Apply | To overlay GF1-T1 on GF21-T0 |
| 10 | Resample transformed image | • Right click green module GF21-T1.txm• Search: Resample transformed image• Interpolation: Lanczos• Mode: extended• Keep other defaults | To re-slice according the alignment of reference T0 |
| Repeat steps 9–10 for each other staining time Ti and then, do the following for all staining times | |||
| 11 | 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 next | To select the same area including only the head for all samples |
| 12 | 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 box | To crop all ROI boxes to the same volume |
| 13 | Orthoslice | • Right click on each green module created in step 10• Search: Orthoslice to create one | To check all orthoslices are matching in all three views (XY, XZ, and YZ) in the main display window |
| 14 | Non-local means filter | • Right click on each green module created in step 10• Search: Non-local means filter• Keep all defaults but make sure Filter: XY planes is selected• Apply | To smooth or reduce noise |
| 15 | Orthoslice | • Right click on each green module created in step 14• Create four orthoslices for each• Set Colormap to standardized range for all | To position the four sampling slices in transversal view at the position of interest in the brain |
| 16 | Line probe | • Right click on each green module created in step 14• Search: Line probe and create four probes• Enter Slice number corresponding to slices chosen in step 15• Sampling options: number of samples 500 | To 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 | |||