Quantifying Cell Shape and Gene Expression in the Shoot Apical Meristem Using MorphoGraphX - Plant Cell Morphogenesis: Methods and Protocols - page 124

3.4 Correcting
Segmentation Errors

Segmentation errors fall into three categories: (1) over-
segmentation when a cell has been divided into many cells, (2)
under-segmentation when two or more cells have been merged
into one, and (3) incorrect segmentation when the boundaries
detected are inconsistent with the data.

1. To correct an over-segmentation error, select the “Pick label”
tool (i.e., the pipette in the mesh toolbar). Press the “Alt” key
and left-click on one of the segmented cells. Then select the
“Fill label” tool (i.e., the bucket in the mesh toolbar), press
the “Alt” key, and left-click on all the labels that need to be
merged. You will see them being merged after each click.
2. To correct an under-segmentation or incorrect segmentation
error, click on the “Label color” button to clear the current
label. Then select the “Fill label” tool (i.e., the bucket in the
mesh toolbar), press the “Alt” key, and left-click on the cells to
clear them. Select the “Add new seed” tool and reseed the
cells. In the case of incorrect segmentation, the watershed can
be guided by seeding the triangles close to the walls to con-
strain the segmentation (Fig. 3c-e ). Use the mesh process
“Segment Mesh” in the “Segmentation” folder to re-segment
the affected cells.

3.5 Co-segmentation

To perform time-course studies of the meristem, we need to make
sure that each cell has the same label in all time points. After seg-
mentation of the fi rst time point, the segmentation protocol is
adapted to reuse the cell labels from the fi rst time point.

1. Load the data into stack 2 and extract the surface of the sec-
ond meristem as before ( see Note 13 ).
2. Load the segmented mesh of the previous time step into
stack 1.
3. On stack 1, tick the “Mesh” check box and select “Cells” in
the drop-down menu next to the check box in the stack 1.
Un-tick the “Surface” check box. You should now see a wire-
frame outline of only the cells from stack 1.
4. Tick the “Surface” check box and the “Label” radio button in
the stack 2.
5. Tick “Stack 1” in the control-key interaction box.

6. Position the camera so as to see both meristems from the side.
7. Holding the control key will allow you to move stack 1 inde-
pendently from stack 2. While holding the control key, move
the stack 1 upward (Fig. 4a ) and move the camera to get a
view from the top.
8. Align some cells of the stack 1 with some cells of the stack 2.
It can be convenient to scale the stack 1 if the cells are signifi -
cantly smaller (Fig. 4b ).