Biology Reference
In-Depth Information
A variety of computational techniques can be used to analyze
high-resolution images of the plant cortical cytoskeleton and quan-
tify their biologically relevant parameters. With a bit of exaggera-
tion, there may be as many, or even more, image analysis methods as
there are publications devoted to the topic, which often hampers
comparison of data from different laboratories. Here we are present-
ing the protocols currently used in our laboratory [
25
], but based to
a large extent on previous work published by others [
26
-
29
], with
the hope to contribute to the standardization of basic approaches.
Some of the quantifi cation methods presented here can be used also
for evaluation of images obtained from fi xed material, e.g., after
antibody staining.
2
Materials and Equipment
Besides specialized equipment and materials listed below, standard
equipment, tools, and consumables for plant in vitro culture will
be required.
The fl uorescent markers listed above are likely to be available upon
request from the authors who published them (
see
also Chapter
6
)
,
either in the form of a plasmid suitable for transformation (which
may be useful for introducing the marker into mutants) or in the
form of seeds of stable transgenic lines. Transgenic
A
.
thaliana
lines carrying GFP-tagged tubulin markers, GFP-TUB6 and GFP-
TUA6, can be obtained also from the public Arabidopsis stock
collections—NASC (
http://arabidopsis.info
) and ABRC (
http://
www.arabidopsis.org
)
—under stock codes N6550 and N6551
(NASC) or CS6550 and CS6551 (ABRC), respectively. Stable
transgenic plants carrying the marker of interest can be then used
to introduce the markers into different genetic backgrounds (e.g.,
various mutants) by crossing.
While, in principle, any fl uorescent cytoskeletal marker can be
used for in vivo imaging, our experience is based mainly on observa-
tions in plant lines carrying two marker constructs expressed under
the viral 35S promoter—GFP-MAP4 [
9
] and GFP-FABD [
30
].
We usually observe roots and cotyledons of young seedlings
(5-8 days after germination) grown on vertical MS plates at 22 °C
with a 16 h-light/8 h-dark cycle (
see
Note 1
). Pharmacological
treatments may be included during cultivation, and seedlings may
be alternatively grown in the dark to achieve etiolation, as etiolated
hypocotyls provide another interesting model especially for TIRF
observation ([
20
];
see
Note 2
). Appropriate controls (e.g., wild
type for mutants, or non-treated plants for pharmacological stud-
ies) have to be included at the same time, since all measurements
can be interpreted only in comparison with data from simultane-
ously grown control plants (
see
Note 3
).
2.1 Plants
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