Time-Lapse Imaging of Developing Meristems Using Confocal Laser Scanning Microscope - Plant Cell Morphogenesis: Methods and Protocols - page 112

Fig. 1 Time-lapse imaging of developing meristems using confocal laser scanning microscope. Cut apices or
NPA-treated seedlings can be used to access the meristem surface, using a water-dipping lens. The images
below are taken from Arabidopsis and adapted from [ 28 ] and [ 8 ]
optical path should not be dissected out. Cut apices are then
transferred to a fresh ACM medium in a clean box that will be
used for imaging. Make sure to keep the apices at a distance
from the boxes edges to allow the displacement of the lens
along the X- , Y- , Z -axis while imaging the meristem. It is usu-
ally better to dissect the apex a few hours before imaging, to
let the apex recover from the wound stress.
2. As soon as naked meristems can be observed in the NPA-
grown conditions, seedlings are transferred to a box contain-
ing Arabidopsis medium without NPA. The transfer can be
done at a later stage too, but we do not recommend it as the
meristem is likely to become smaller as the stem elongates and
as a longer stem will increase the chances of having random
meristem movements while imaging it under the CLSM. The
whole plantlets are stuck to the medium with a generous
amount of lukewarm melted 0.5 % agarose (Fig. 1 ). Using a
binocular, make sure that the meristem is properly oriented. In
comparison with cut apices from soil grown plants, the integ-
rity of the whole plant is preserved when using NPA-grown
seedlings. However, NPA may induce artifacts and the size of
the meristem is much smaller.