Manual Segmentation
===================

Description
-----------

The following workflows should work both with ImageJ and Fiji,
after these have been correctly set up with
the OMERO plugin for Fiji/ImageJ.

Using the User Interface of the OMERO plugin, we will show:

-  How to connect to OMERO using the OMERO plugin for Fiji/ImageJ.

-  How to open an image from OMERO.server into Fiji/ImageJ.

-  How to manually segment an image opened from OMERO in Fiji/ImageJ
   using the plugins *Auto Threshold* and *Analyze particles* in Fiji/ImageJ.

-  How to record this workflow in Fiji/ImageJ, using the *Recorder* plugin, for later use.

Setup
-----

-  Fiji has been installed on the local machine with the
   OMERO.insight-ij plugin. The installation instructions can be
   found at :doc:`installation`.

Resources
---------

-  Samples images from the Image Data Resource (IDR) `idr0021 <https://idr.openmicroscopy.org/search/?query=Name:idr0021>`__.

Step-by-step
------------

#. Launch Fiji/ImageJ.

#. Go to *Plugins > OMERO > Connect* To OMERO. This will show a login
   screen where you can enter the name of the server to connect to,
   the username and password. The OMERO plugin will allow you to
   browse your data in a similar manner to OMERO.web.

#. In the OMERO login dialog, click the wrench icon\ |image0| and then
   add the server address in the dialog. By default, only "localhost"
   is listed. Click on the *plus* icon to add a new line to the list
   and type into the line the server address.

#. Click Apply.

   .. image:: images/manual2.png

#. Enter your credentials and click *Login*.

#. Browse to the Project **idr0021**, open any Dataset and double-click once
   on an Image to open it in Fiji. Bio-Formats is used to view the
   Image.

   - Make sure to select *View stack with: Hyperstack* in the *Bio-Formats
     Import Options* dialog.

   - Note that each plane will be transferred from the server to the
     client machine so this may take a few moments.

#. To open the Recorder, go *Plugins > Macros > Record...*, select
   Macros to record the actions. The steps will then be used in
   the Scripting workflow.

#. Convert floating-point pixel-type to 8-bit using *Image > Type > 8-bit*.

#. Go to *Image > Adjust > Auto Threshold*, to open the *Auto Threshold* dialog:

   - Select *MaxEntropy* for the *Method* parameter.

   - Check the checkbox *Stack*.

   ..

   |image2|\ |image3|

#. Click *OK*.

#. Then open *Analyze > Analyze Particles...*.

#. In the dialog

    - Set *Size* to *10-Infinity* and check *Pixel units*.

    - Check the following checkboxes:

       i.   *Display results*

       ii.  *Clear results*

       iii. *Add to Manager*

    - Click *OK* then *Yes* in the popup dialog indicating asking to
       *Process all X images?*.

   ..

   |image4|

#. To save the thresholded Image back to OMERO with the generated ROIs and the measurements:

    -  Select *Plugins > OMERO > Save Image(s) to OMERO*.

    -  Create a New Dataset for the image

       i.   Click the *New...* button next to the selection box on the
            Dataset row.

       ii.  In the dialog that pops up, enter a name and a description
            (optional).

       iii. Click Create.

    -  The newly created Dataset will automatically be selected.

    -  Click *Add to the Queue* then *Import*.

    -  Go to OMERO.web and check that the measurements have been saved
       in a CSV file and attached to the Images. The attachment can
       then be downloaded at any time.

#. To save the recorded macro to OMERO.server:
    -  In Fiji, find the Recorder window and click "Create". A new window will pop up with the macro you just recorded. Verify the macro on a new image. Once the verification succeeds, just close the macro window and in the dialog which pops up select "Yes" to save the macro. Save the macro locally e.g. "your-macro-name.ijm".
    -  In OMERO.web, select the dataset you would like the macro to be run on and attach the "your-macro-name.ijm" macro you just recorded as file attachment to that dataset. The attachment can then be downloaded at any time. Also, the attached macro can be used by Fiji scripts - see example on `threshold_scripting.html <https://omero-guides.readthedocs.io/en/latest/fiji/docs/threshold_scripting.html>`_.

.. |image0| image:: images/manual1.png
   :width: 0.24105in
   :height: 0.24105in
.. |image2| image:: images/threshold1.png
   :width: 3.33854in
   :height: 3.32695in
.. |image3| image:: images/threshold2.png
   :width: 2.25521in
   :height: 2.69626in
.. |image4| image:: images/threshold3.png
   :width: 2.57813in
   :height: 2.5947in