**********************
Segmenting cells in 2D
**********************

.. figure:: images/segmentation-2d-cover.*
    :alt: Shoot apical meristem of A. thaliana, with microtubules
    :align: center
    :figwidth: 85%
    :width: 100%

    Shoot apical meristem of A. thaliana, with microtubules

.. _dataset: http://www.botany.unibe.ch/deve/lithographx/Microtubules.zip

In this section, we are going to see how we can extract the 2D shape of cells in
images of the shoot apical meristem of *Arabidopsis thaliana*, and see how we
can use a marker of cortical microtubule to compute their orientation based on
the method published in [Boudaoud2014]_. To start, download this `dataset`_,
provided by Dr. Agata Burian.

To segment the cells in 2D, we need to proceed in two phases:

#. Extract the surface of the meristem
#. Segment the cells on that surface

Extracting the surface of the meristem
======================================

See figure :num:`segmentation2d-fig-seg` A-D.

#. Blur the stack slightly:

    .. LGXProcess::
      :name: Gaussian Blur Stack
      :folder: Filters
      :type: Stack

      X Sigma (μm): 0.5
      Y Sigma (μm): 0.5
      Z Sigma (μm): 0.5

#. Extract the surface, using all the default arguments:

    .. LGXProcess::
      :name: Edge Detect
      :folder: Morphology
      :type: Stack

#. Erase the bumps due to dead cells by hand, using the ``Pixel Edit`` tool
#. Extract a coarse surface:

    .. LGXProcess::
      :name: Marching Cubes Surface
      :folder: Creation
      :type: Mesh

      Cube size (μm): 3

#. Select the bottom and sides with the selection tool: make sure the
   ``Mesh`` check-box is ticked and ``View`` is on ``Selected``. Then,
   orienting properly the meristem, press the ``Alt`` key and click and
   drag a rectangle to select the vertices to remove. You can extend the
   selection by pressing the ``Shift`` key while clicking. For a demonstration
   of what needs to be done, look at this video.
#. We now need to refine the surface. For this, we will alternate smoothing and
   refining the mesh. Check the terminal for the number of vertices after
   subdivision.

    .. LGXProcess::
      :name: Smooth Mesh
      :folder: Structure
      :type: Mesh

      Passes: 3

    .. LGXProcess::
      :name: Subdivide
      :folder: Structure
      :type: Mesh

.. youtube:: http://www.youtube.com/watch?v=ZbTaYITkObg
    :width: 512
    :height: 395

.. _segmentation2d-fig-seg:

.. figure:: images/segmentation-2d-segmentation.*
    :alt: 2D segmentation of the meristem
    :align: center
    :figwidth: 85%
    :width: 100%

    2D Segmentation of the meristem

Segment the cells
=================

See figure :num:`segmentation2d-fig-seg` E-F.

#. First, make sure the PI channel, which should be the Main Stack 1 is the
   active stack. In the Main tab, the ``Stack 1`` tab should be the selected
   tab, the ``Main`` store should be selected and the ``Work`` store not.
#. Then, project the signal onto the surface. You can play with the mim and max
   distances to see what happens when they change.

    .. LGXProcess::
      :name: Project Signal
      :folder: Signal
      :type: Mesh

      Min Dist (μm): 1
      Max Dist (μm): 1

#. Now, we are going to segment the cells visible in the signal.

    .. LGXProcess::
      :name: Auto-Segmentation
      :folder: Segmentation
      :type: Mesh

      Update: Yes
      Normalize: No
      Blur Cell Radius (μm): 1
      Auto-Seed Radius (μm): 2
      Border Distance (μm): 1
      Combine Threshold: 1


#. After a while, you will see outline of segmented cells. In the Main
   tab, select ``Labels`` as representation for the surface. And using the
   2D bucket, erase the cells above the areas where the staining didn't
   work.

Micro-tubule orientations
=========================

.. figure:: images/segmentation-2d-orientation.*
    :alt: Extracting the orientation of the microtubules
    :align: center
    :figwidth: 85%
    :width: 100%

    Extraction of the microtubules orientation.

#. Load the MBD stack: just drag and drop the file on the LithoGraphX window.
#. Project the new signal onto the surface. Select a range from *-1 µm* to *1
   µm*. You can try different values and see which provide the best contrast.
#. Compute the fibril orientations:

    .. LGXProcess::
      :name: Compute Fibril Orientations
      :folder: Cell Axis/Fibril Orientation
      :type: Mesh


#. You can also adjust the way the orientation is displayed:

    .. LGXProcess::
      :name: Display Fibril Orientation
      :folder: Cell Axis/Fibril Orientation
      :type: Mesh

.. [Boudaoud2014] Boudaoud, A.; Burian, A.; Borowska-Wykręt, D.; Uyttewaal, M.;
    Wrzalik, R.; Kwiatkowska, D. and Hamant, O. **FibrilTool, an ImageJ plug-in to
    quantify fibrillar structures in raw microscopy images**. *Nature Protocols*,
    2014, 9, 457-463