(part 1) Step 3: finding orientation (basal foot)
Again, for this step, make sure that the Correlescence plugin version >= 0.0.7 is installed on your FIJI.
At this point it is assumed that step 2 was performed and data extracted from BigTrace sits in the s2_extracted subfolder inside one proteinName1_proteinName2_decov_001 folder.
Open FIJI and run s3_find_orientation_batch_scale.ijm macro. The window with parameter choices is going to appear, where all but one parameter are described here. The one extra parameter that needs to be specified is Reference channel = the number of the channel with total protein (panExM) stain;
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.
NB: While this macro is running, it is a good idea to 'leave Fiji and PC alone', i.e. do not perform any other tasks, since it will break the execution of the macro.
Sometimes some errors may appear in the Console log window, they can be ignored, the data will be saved.
In the folder specified in the last step, the macro will create a new s3_rotated_XY[scale]_Z[scale] subfolder.
(if XY or Z scales are equal to 1, these suffixes will be omitted)
Inside this subfolder, there would be tiff volumes that are rotated in a way that the detected basal foot is pointing upwards.
This data will be used for the next step.
To create XY and XZ max projections of these files, you can run generate_xy_xzyz_proj.ijm macro.
In addition to that, the macro will create a detected subfolder inside.
In this folder, for each file there is a 3-channel image, where:
- channel 3 contains maximum intensity projection of the reference channel (NOT ROTATED!);
- channel 2 contains the line marking the detected position of the basal foot;
- channel 1 contains max intensity projection image enhanced using tubeness filter.
Here is an example of UnequalTiffs plugin "Montage" command for the illustration
The next step is (part 1) Step 4: averaging BB per cell.
First, macro takes only total protein stain channel z-stack
and builds its XY maximum projection (image below, left).
To get rig of background and enhance lines, we apply tubeness filter (FIJI)
with the sigma equal to the XY SD of the PSF (image below, right).
Next, we determine the center and diameter of the ring
using the same procedure as in step 1.
Then we create a template of radial line segments with an angle step of 1 degree.
They emanate from the center with a starting point outside of the ring image,
spanning outwards for half the ring diameter (image below, top middle panel).
It is a way to simulate the appearance of basal feet.
We calculate the maximum of the normalized cross-correlation (NCC) between the images
of line segments and tubeness filtered ring image, excluding any spatial shift (bottom panel).
This gives us the angle of basal feet.
The volumes are rescaled by provided factors and rotated in a way,
so the basal feet points upwards.