Biology Reference
In-Depth Information
2
Materials
2.1 Confocal Images
For this protocol, we require confocal images of a shoot apical
meristem with the cell wall or cell membrane labelled in some way.
For example, the samples in the fi gures used propidium iodide (PI)
staining. Optionally other markers, localized on the plasma mem-
brane or inside the cell, may be collected in other channels [
1
,
2
].
When acquiring images for the purpose of 3D reconstruction
with MorphoGraphX, the following principles should be followed.
1. Images should be acquired in 16 bits per pixel: this is the
native image format of MorphoGraphX. All the processing
will be done in 16 bits, and the extra information is often cru-
cial for correctly reconstructing areas of the image with low
intensity. If 16 bit collection is not available on your micro-
scope, use 12 bit or the highest available setting.
2. Use cubic voxels if possible (i.e., the z-step is equal to the
resolution in
x
and
y
).
3. Add planes (i.e., reduce the z-step) rather than using line or
frame averaging. For the same amount of exposure to the
laser, you will get extra information on the z position.
4. Adjust the offset and gain of the sensor to maximize the
dynamic range of your image (
see
Note 1
).
5. Collect extra Z-slices above and below the area of interest of
the meristem. The fi rst and last slices should not contain any
useful signal: they are used to limit the size of the meristem
along the
z
-axis. This will ensure you have the correct shape
for the whole meristem, including the tip.
6. Most microscopes save their images in a proprietary format.
MorphoGraphX can read a set of standard fi le formats but
works best with single TIFF stacks as written by ImageJ or FiJi
[
4
-
6
]. Load the images using ImageJ with the LOCI plug-in
[
7
] and save it as a single TIFF stack (
see
Note 2
).
You will need a computer with a recent nVIDIA graphics card that
supports CUDA. A card with 2 Gb or more dedicated video mem-
ory is recommended. The computer itself should have at least 8 Gb
of main memory, and a multi-core CPU is benefi cial for some
processes.
2.2 Computer
3
Methods
Once the image is acquired, the fi rst step is to visualize it. The
stack should be in a single TIFF fi le, with only one time point and
channel per fi le.
3.1 Visualization
and User Interaction
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