Quantifying Cell Shape and Gene Expression in the Shoot Apical Meristem Using MorphoGraphX - Plant Cell Morphogenesis: Methods and Protocols - page 120

1. Launch MorphoGraphX. You should see a single window, with
a black central area (the visualization zone), some menus and
toolbars, and a right panel with three tabs (Fig. 1 ). You will also
see a terminal window opened with some text ( see Note 3 ).

2. Drag the fi le containing the fi rst time point of your meristem
and drop it into the visualization area of MorphoGraphX. You
should see the volumetric image appear in the visualization
area (Fig. 1b , see Note 4 ). You should be able to rotate and
translate the image by using the left and right mouse buttons.
The wheel can be used to zoom. A complete description of
camera manipulations can be found in the online help of the
software, accessible either from the “Help” menu or by press-
ing the “h” key.
3. Assess the quality of the acquisition ( see Note 6 ): the meristem
should be “complete” (i.e., it must not be cropped); the cells
of interest must be entirely visible; there should not be holes
or spots in the image (i.e., no leak of the wall marker inside the
cells and no large interruption of the marking of the wall).

Since the meristem is a dome-shaped organ with considerable sur-
face curvature, there is no fl at projection of the cells that would
conserve their geometry. Instead, the shape of the meristem's sur-
face is extracted, and the signal projected onto this curved surface
for further processing.

3.2 Extracting
the Meristem Surface

1. Use the stack process “Gaussian Blur Stack” in the “Filters”
folder to smooth out the noise ( see Note 7 ).
2. If required, remove any unwanted objects. Use the voxel edit-
ing tools in erase mode ( see Note 8 ) to remove any object that
is not part of the meristem.
3. Save the stack to a new fi le, and copy it to the main store with
the stack process “Copy to Work to Main Stack” in the “Multi-
stack” folder.
4. Use the stack process “Edge Detect” in the “Morphology”
folder to obtain a fi lled-in stack of the meristem shape ( see
Note 9 ). The threshold determines what light intensity (rang-
ing from 0 to 65,536) is considered to be the surface. If there
are large holes in the shape, then repeat the edge detection
with a lower threshold. If there are only small holes, use the
stack process “Fill Holes” to fi ll them in. It is important that
this step is done well. To check that the fi lled-in shape matches
the original stack well, turn on the main store to make it visi-
ble, and then use the clipping plane to compare the two stores
( see Note 5) . The top of the data should match the top of the
extracted shape (Fig. 2a ). If there is not a good match, try
again with a higher threshold. Too high a threshold will result
in holes.