(part 1) Step 2: extracting basal bodies
For this step, make sure that you have the BigTrace plugin version >= 0.5.1 is installed on your FIJI.
At this step, we will use .csv files (tables with XYZ coordinates) from the previous step
to create 3D ROIs for BigTrace plugin and extract volumes around BBs.
It is assumed they are located in the s1_detected subfolder of an arbitrary proteinName1_proteinName2_decov_001 folder.
- Open FIJI and run s2_cvs_extract_BigTrace_rois.ijm macro and a parameter dialog will appear.
- The first parameter is the pipe's diameter of extraction around the centerline of a BB in pixels.
- The second parameter describes a smoothing window size (in pixels), applied to the coordinates of the centerline.
After clicking OK, a dialog with the choice of output storage folder will appear.
Please select the corresponding proteinName1_proteinName2_decov_001 folder with tif file and ROIs file, as described here.
In the proteinName1_proteinName2_decov_001 folder (specified in the last step),
the macro will create s2_extracted subfolder.
A set of tif files should appear in this subfolder with names in the format of ROINAME_straight.tif.
Those are "straightened" versions of each BB.
Notice, that the voxel size of these volumes becomes isotropic,
i.e. Z-step is equal to XY pixel size.
There is an extra BT_rois subfolder inside it with 3D ROIs in BigTrace format, used for extraction from the original tif volume.
You can make XY XZ maximum intensity projections of these volumes using generate_xy_xzyz_proj.ijm macro.
These files (or generated max projections) can be inspected individually or using UnequalTiffs plugin.
Next step is (part 1) Step 3: finding orientation (basal foot)
In the previous step we had XYZ centerline coordinates of all detected BBs.
The macro converts each of them to the linetrace 3D ROI of Bigtrace.
And saves them in the corresponding BigTrace format.
It looks like this:
where the red lines represent detected centerlines.
Using centerlines, it is possible to build a "pipe" around each of the BB,
with a specified diameter of extraction, as shown here:
For that, we use the rotation minimizing frame algorithm.
Once the pipe is constructed, it is possible to "straighten"
volumetric intensity data inside it using the specific BigTrace function.
The details on geometry behind it and intensity interpolation are provided here.
