4.5. Segmenting cells in 2D

Figure 1: Shoot apical meristem of A. thaliana, with microtubules

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:

1. Extract the surface of the meristem
2. Segment the cells on that surface

4.5.1. Extracting the surface of the meristem

See figure 2 A-D.

1. Blur the stack slightly:

   Process	[Stack]Filters/Gaussian Blur Stack
   Parameter	Value
   X Sigma (μm)	0.5
   Y Sigma (μm)	0.5
   Z Sigma (μm)	0.5

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

   Process	[Stack]Morphology/Edge Detect

3. Erase the bumps due to dead cells by hand, using the Pixel Edit tool

4. Extract a coarse surface:

   Process	[Mesh]Creation/Marching Cubes Surface
   Parameter	Value
   Cube size (μm)	3

5. 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.

6. 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. You really should ends with around 250‘000 vertices.

   Process	[Mesh]Structure/Smooth Mesh
   Parameter	Value
   Passes	3

   Process	[Mesh]Structure/Subdivide

Figure 2: 2D Segmentation of the meristem

4.5.2. Segment the cells

See figure 2 E-F.

1. 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.

2. Then, project the signal onto the surface. You can play with the mim and max distances to see what happens when they change.

   Process	[Mesh]Signal/Project Signal
   Parameter	Value
   Min Dist (μm)	1
   Max Dist (μm)	4

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

   Process	[Mesh]Segmentation/Auto-Segmentation
   Parameter	Value
   Update	Yes
   Normalize	No
   Blur Cell Radius (μm)	1
   Auto-Seed Radius (μm)	2
   Border Distance (μm)	1
   Combine Threshold	1

4. 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.

4.5.3. Micro-tubule orientations

Figure 3: Extraction of the microtubules orientation.

1. Load the MBD stack: just drag and drop the file on the LithoGraphX window.

2. 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.

3. Compute the fibril orientations:

   Process	[Mesh]Cell Axis/Fibril Orientation/Compute Fibril Orientations

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

   Process	[Mesh]Cell Axis/Fibril Orientation/Display Fibril Orientation

[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