4.3. Segmenting Cells in 3D

Figure 1: Shoot apical meristem of A. thaliana, segmented in 3D

In this section, we are going to see how we can extract the 3D shape of cells in images of the shoot apical meristem of Arabidopsis thaliana, and see how we can use an extra channel with the segmentation. To start, download this dataset, provided by Dr. Agata Burian.

4.3.1. Loading the data

For this tutorial, you can start either by loading the file Arabido_DR5-YFP_pi.tif or Arabido_DR5-YFP_pi_cleaned.tif.

4.3.2. Cleaning the data

If you have loaded Arabido_DR5-YFP_pi.tif, you will see in addition to the shoot apical meristem a few lateral organs, including those partially removed to acquire the image. To extract only what we want to see, we need to clean the image.

1. Copy the stack to the work store. At the same time, we will autoscale the stack’s intensity. This is an important step as it makes to rest of the process much easier.

   Process	[Stack]Filters/Autoscale Stack

2. Select the pixel edit tool. In the View tab make sure Fill checkbox in the Stack Editing area is not checked. Then, pressing the Alt key, you should see a circle above the drawing area. This circle marks the cylinder that will get erased. Click to erase.

3. When you are happy with your cleaning, save the stack!

Note

If the area to erase is too large, nothing will change until you release the mouse button.

Hint

You can also do the cleaning on the segmented stack. In this case, another useful tool is the fill volume tool. Ensure no label is currently selected by clicking on the current label button. Then click on the labels you want to erase.

4.3.3. 3D Cells Segmentation

1. Make sure the stack is in the Main store.

2. Blur the stack

   Process	[Stack]Filters/Gaussian Blur Stack
   Parameter	Value
   X Sigma (μm)	0.3
   Y Sigma (μm)	0.3
   Z Sigma (μm)	0.3

3. Optionnally, use the Sieve filter. It can be quite slow, but it really improves the segmentation.

   Process	[Stack]Morphology/SieveFilter
   Parameter	Value
   Type	Median
   Size (μm²/μm³)	4

4. Segment the stack:

   Process	[Stack]ITK/Segmentation/ITK Watershed Auto Seeded
   Parameter	Value
   Level	1500

5. Remove external cells:

   Process	[Stack]Segmentation/Erase at Border
   Parameter	Value
   Distance (μm)	0
   X	Yes
   Y	Yes
   Z	Yes

6. Extract the cell shapes:

   Process	[Mesh]Creation/Marching Cubes 3D
   Parameter	Value
   Cube Size (μm)	1
   Smooth Passes	3

Hint

Use the clipping planes to scan through the volume and make sure the segmentation worked correctly in the depth of the tissue.

4.3.4. 3D Signal Quantification

Figure 2: Signal quantification on 3D mesh

1. Load the YFP channel in the work store of the stack 1. To do that, the simplest is to drag&drop the file onto the work store area.

2. Launch the Heat map mesh process:

   Process	[Mesh]Heat Map/Heat Map

3. In the dialog box, select the Volume heat map type and the Total signal visualization and make sure Signal average is not ticked.

The result is, per cell, the total amount of signal present in the YFP channel.

Note

This is not sufficient for a proper signal quantification. This is only qualitative. For a proper quantification of the signal you need a reference channel.