Visualizing and Quantifying the In Vivo Structure and Dynamics of the Arabidopsis Cortical Cytoskeleton Using CLSM and VAEM - Plant Cell Morphogenesis: Methods and Protocols - page 92

3.7 Kymograph
Construction from
VAEM Image Series

Kymographs can be used to visualize aspects of microfi lament and
microtubule dynamics that are not easily observed in the video
sequences.

1. Open the *.lif fi le generated by VAEM by dragging it onto the
ImageJ window; use the “open as hyperstack” option in the
dialog box, and select the desired image to evaluate.
2. Defi ne a specifi c line length by ROI selection
(Plugins > ROI > SpecifyLine) and locate the line across a repre-
sentative area of the image (across a well-focused part of a cell).

3. Generate the kymographs using the plug-in Multiple
Kymograph (Plugins > MultipleKymograph with linewidth: 3).
4. The image generated shows velocity, movement, and different
phases of microfi lament or microtubule turnover ( see Note 13 ).

4 Notes

1. Use the culture media and protocols established in the labora-
tory; any medium and culture setup that allows easy removal
of intact seedlings from plates should work. Alternatively,
seedlings may be grown on a medium-covered slide surface in
situ to avoid disturbance of, e.g., root hairs.
2. Any tissue that can be positioned fl at towards the cover slip
surface ought to be accessible to CLSM and VAEM; especially
for the latter, tight contact with the cover slip is critical. Leaves
of glabrous mutants and petals may be especially worth
exploring.
3. Ideally, the measurements should be done at least in a single-
blind manner to eliminate observer bias (i.e., the person per-
forming quantitative image analysis should not know which
image series belongs to which genotype or experimental
treatment).
4. In our case, both microscopes are in the inverted confi gura-
tion. For an upright microscope, sample preparation may have
to be modifi ed.
5. This software exists also in versions for other operation sys-
tems such as Linux.
6. Take care to treat all the seedlings equally, since mechanical
stress may elicit modifi cation of cytoskeletal organization and
dynamics during the plant manipulation, media exchange, or
even cover slip placement. For longer observation, edges of the
cover slip may be sealed with nail polish, but this is usually not
necessary. Do not use too much water, as the cover slip should
be held in place by capillary forces rather than move around on
excess liquid (this is easier to achieve with large cover slips).