Biology Reference
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7. If working carefully, live seedlings can be recovered from the
slide after observation and transferred ex vitro for further cul-
tivation, but do not expect 100 % survival.
8. In case of high autofl uorescence background, the window
should be narrowed (i.e., longer wavelengths should be cut off
in the Beam Path settings).
9. Cytoskeletal structure and dynamics varies dramatically with
anatomical location; therefore, imaged cells should be located
consistently (e.g., at the bottom of the root tip elongation
zone or in the middle of the cotyledon).
10. Maximum intensity projections from the serial optical sections
can serve also for determining cytoskeletal fi lament orienta-
tion. Reference [ 28 ] describes a procedure employing noise
reduction, conversion of the images to binary, and skeletoni-
zation. The resulting skeletonized image is used to evaluate
cytoskeletal architecture in guard cells of the stomata. Mean
angular difference between microfi lament pixel pairs and the
nearest pixel pairs of a specifi c cell edge (the stomatal pore) is
used as a measure of microfi lament orientation. The proce-
dure can be checked by obtaining synthesized images.
Analogously, microtubule orientation can be determined with
respect to the cell's specifi c axis, e.g., the longitudinal axis of
the hypocotyl [ 35 ].
11. It is recommendable to maintain a constant location/direc-
tion of the sampling line within a cell, e.g., along the longitu-
dinal axis in case of rhizodermis.
12. Additional data about actin assembly rates, fi lament origin, and
severing frequency can be obtained by the analysis of stochastic
dynamics as described in ref. [ 20 ], where actin fi laments are
tracked manually through the time-lapse stack of images and
different actin dynamic parameters are estimated by overlap-
ping images or monitoring breaks along the fi lament over time.
13. Choose the length and location of the sampling line consis-
tently (e.g., parallel to the longitudinal axis in roots and
hypocotyls; see ref. [ 29 ]). While 1 min is usually enough to
document microfi lament dynamics, in the case of the less
mobile microtubules 2 min provides a more informative result.
Acknowledgments
This work has been supported by the GA
R P305/10/0433 proj-
ect. We thank Boris Voigt and Richard Cyr for transgenic
Arabidopsis lines; Ond
Č
ej Horváth, and Aleš
Soukup for expert microscopy advice; and Marta
ř
ej Šebesta, Ond
ř
Č
adyová for
technical assistance.
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