Error message set position slice number ROI manager

[mmjulkowska]

I am trying to run SPIRO Assays for Root Growth, and I get "germination data" all right from it - but when it comes to actually tracing the root growth - I run into the following error:

[mmjulkowska]

Here is the Debug window screenshot

[jiaxuanleong]

Hi! Thanks for submitting an issue. May I know what version of ImageJ you are running? You can find this at Help > About ImageJ.

The unrecognized command could be due to an old ImageJ version since RoiManager.setPosition was only introduced in 1.53c. Could you please try running the macro using a clean install of FIJI ImageJ v1.53f with only the required plugins TurboReg and MultiStackReg). At the time of writing ImageJ v1.53f has been confirmed to run smoothly i.e. recognizing all commands used in the SPIRO macros. You can get this version from the FIJI archive

[mmjulkowska]

Thanks. I have downloaded the correct version - now the software is stuck here:

[jiaxuanleong]

Hi! Was everything fine during the step where a dialog box pops up with "Please delete any ROIs that should not be included into analysis, e.g. objects wrongly recognized as seeds. Unrecognized seeds can also be added as ROIs."? Did the ROI Manager display properly and you could add/change ROIs?

If that went fine, then ROI Manager did not save the file. Can you verify this for me by going to your folder containing plate 1 Col-0 and checking the .zip file does not exist?

I noticed also in your screenshot that you have seedlings already in your first time point. The automatic detection of seeds does not work then and you would have to manually select the "root starts" i,e the top of the root and add them as ROIs. I have attached an image to show what I mean.

[mmjulkowska]

Thanks again - the zip file indeed does not exist - see the screenshot below:

Moreover - this message is popping up before I had a chance to select any ROI. Once I did select my ROI - I dont have any message box to click on to proceed to the next step:

Regarding roots on the 1st image - we are interested in root growth - not germination. Since we are transplanting/treating the plants at 4 days old stage, we do not have the images containing the seed at the beginning of the experiment.

[jonasoh]

@mmjulkowska Just a heads up that we're currently using the germination data for root growth normalization downstream, so the provided R scripts will not work using your current setup. The data from the macro can still be used for analyses though, but you may need to tweak the scripts or do your own custom analysis.

In our experience, we needed to normalize root growth to germination occurrence, since the variation within the group would otherwise be too large when some seeds would get a "head start". Therefore we set the zero timepoint per-seedling as the timepoint where germination is detected.

This may not be needed if you're only interested in root growth rates though, but beware that the scripts will not work for your use case out of the box.

[mmjulkowska]

Thanks @jonasoh for the heads-up - we can do/adjust the R analysis downstream no problem. Once we have the R script that is working for us - we would be happy to share it with y'all :)

RE variation: Since we transfer the seedling to our treatment plates - we are already reducing the germination variation by transferring only the seedlings of the approximately same size.

[jonasoh]

Sounds good! We'd very much like to see others building their own pipelines based on the SPIRO hardware :)