Live Dead then Neurite Outgrowth
Assistance needed on segmentation overlays in journal output.

I've made this journal that runs Live Dead and then uses live nuclei in the Neurite Outgrowth module. File attached. As far as I can see I have enabled saving of the segmentation overlays to the database, but when I call up the images afterwards there are no overlays present. This is my first journal so I'm still getting used to things.

What do I need to change to get the segmentation overlays into the database? Do I need to first display the resulting images?

Attached Files
.jnl   Neurite Outgrowth Live Dead.JNL (Size: 9.79 KB / Downloads: 1)
Hi Rich

When you run a module via a journal the segmentation is not being saved to the database. An alternate method would be to use Custom module analysis in which the segmentation will be saved and it allows you to combine different modules as well.
Another alternative is to configure the journal to save the segmentation masks to image files in a folder on the computer.
Thanks both for your responses.

I prefer the overlays to be saved to the database, so that I can review how effective the segmentation has been. If they are saved to folders this will be more difficult and time consuming. My alternative is to run the journal manually on each site I want to check as I think this retains the overlays visually.

I don't understand why these features are not included. Another thing I can't do (in v5 at least) is get the application module measurements from the CME (e.g. neurite outgrowth). I can only use it for masks. So Sylvia's alternative method doesn't work for me.
We do have both of these items on our software requests list:
- allow journals to save segmentation masks to the database
- make application module measurements available as outputs from CME (especially neurite outgrowth)

However, since we do not have those available today, let's see if we can find another approach that will fit your workflow.

It looks like you are using FITC as the Live marker. You may well have already tried this, but sometimes using FITC itself for the nuclear mask in Neurite outgrowth may be helpful.

Here are some other possible approaches involving journals.
1. Run a journal during acquisition to overwrite the Live Dead result image into a 3rd dummy wavelength which can then be used as your nuclear source when running Neurite Outgrowth.
2. Similar to #1 but done post-acquisition - use a journal to produce the Live Dead result image and FITC image into a format that can easily be imported back as a new plate in the database for analysis with Neurite Outgrowth.
3. Run your journal analysis and save the segmentation masks as overlays on top of the original image to a folder, so that you can toggle the mask on and off the original images (e.g. DAPI in blue, FITC in green, segmentation in red). These overlays could be saved either as individual files which you can flip through in Windows, or as a stack/multi-plane TIFF that you can open in MetaXpress and scroll through.

Let us know if you think any of these workflows would be helpful, and we can help you with the journaling as needed.
Hi Paula,

Good to hear that those requests are in the list!

I did try doing neurite outgrowth without the nucleus option at all, but I haven't tried using the FITC for both parts of the analysis. I'll look into that.

I like the idea of your suggestion number 1, for future plates. I'll need to run my journal without the neurite outgrowth part, can you suggest a method to get the image saved?
Can this only be done during acquisition, or could I also take this approach to save the segmentations to dummy wavelengths post-acquisition?

Thanks for the help.
Hi Rich,

Here is an example journal for #1.

1) Identify a wavelength to use as your dummy wavelength.  This can either be a wavelength not used in your experiment (e.g. Cy5) or you can create a fake wavelength in Devices > Configure Illumination (or Control > Devices > Configure Illumination).  Name new setting something like "Live Dead Result", no shutters or light sources need to be enabled, and click "Add/Replace".

2) Set up Plate Acquisition with 3 wavelengths: DAPI, FITC, and your 3rd wavelength which must be at W3 (DAPI and FITC must be acquired prior to the dummy wavelength).  Set the exposure time for W3 to 1 ms.

3) If in MX6, on the Acquisition tab, enable the option to Run Journals During Acquisition.

4) On the Journal tab, select the "After Each Image" option and select this journal.  If this is the only journal being used, you should not need to enable the option to "Prevent asynchronous hardware moves", which will slightly slow down the acquisition.  

5) On the Display tab, make sure the option to "Display images during acquisition" is enabled.

6) Run the Plate Acquisition.

For post-acquisition:
You cannot add additional wavelengths to an existing plate in the database.  I would recommend my #2 above, where we run a journal to create a new set of images that can be imported into the database.  This could either just be FITC + Live Dead result, or DAPI + FITC + Live Dead result, depending on your preference. 
Let me know what you think.

Attached Files
.jnl   Live Dead_After Each Image.JNL (Size: 4.87 KB / Downloads: 3)

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