such that the confocal volume will likely accommodate further
growth of the tissue.
2. Another consideration during time-lapse imaging is dehydration. Since water evaporates during imaging, pre-fill the dish
with an amount of water that will last for the entire experiment.
It is not advisable to top-up the water level during the course of
the experiment because the sample will likely move.
3. Reducing photodamage and bleaching are key aspects for a
successful time-lapse experiments. Therefore optimize laser
power, size of the z-stack, and frequency of the z-slices/size
of the optical sections, as well as the frequency of the time
intervals as necessary.
4 Notes
1. Imaging of seedling under a confocal microscope is generally
not performed in sterile conditions. This might result in the
growth of fungi or bacteria. To avoid contaminants, Carbenicillin (300 mg per liter) can be added to the growth medium
and seedlings can be transplanted to new dishes frequently.
2. Seedlings are very sensitive to growth conditions and they can
develop at different speeds. Therefore, the frequency of imaging appropriate for specific developmental stages and different
plants may need re-assessment frequently.
3. We found that it is easier to dissect cotyledons without damaging the seedlings when they are young (as mentioned
2.5–3 days after stratification). Moreover, to image the VM
later we suggest removing older leaves gradually as they form
and grow.
4. When adding water, air bubbles might be trapped around and
between the leaves and thereby impede imaging. To remove
them we gently pipette water up and down (being careful not
to touch the meristem).
Acknowledgments
We acknowledge the Australian Research Council and European
Research Council for support.
References
1. Fernandez R, Das P, Mirabet V, Moscardi E,
Traas J, Verdeil JL, Malandain G, Godin C
(2010) Imaging plant growth in 4D: robust
tissue reconstruction and lineaging at cell
resolution. Nat Methods 7(7):547–553.
https://doi.org/10.1038/nmeth.1472
2. Bhatia N, Bozorg B, Larsson A, Ohno C,
Jonsson H, Heisler MG (2016) Auxin acts
Confocal Microscopy of Arabidopsis Seedlings
301
growth of the tissue.
2. Another consideration during time-lapse imaging is dehydration. Since water evaporates during imaging, pre-fill the dish
with an amount of water that will last for the entire experiment.
It is not advisable to top-up the water level during the course of
the experiment because the sample will likely move.
3. Reducing photodamage and bleaching are key aspects for a
successful time-lapse experiments. Therefore optimize laser
power, size of the z-stack, and frequency of the z-slices/size
of the optical sections, as well as the frequency of the time
intervals as necessary.
4 Notes
1. Imaging of seedling under a confocal microscope is generally
not performed in sterile conditions. This might result in the
growth of fungi or bacteria. To avoid contaminants, Carbenicillin (300 mg per liter) can be added to the growth medium
and seedlings can be transplanted to new dishes frequently.
2. Seedlings are very sensitive to growth conditions and they can
develop at different speeds. Therefore, the frequency of imaging appropriate for specific developmental stages and different
plants may need re-assessment frequently.
3. We found that it is easier to dissect cotyledons without damaging the seedlings when they are young (as mentioned
2.5–3 days after stratification). Moreover, to image the VM
later we suggest removing older leaves gradually as they form
and grow.
4. When adding water, air bubbles might be trapped around and
between the leaves and thereby impede imaging. To remove
them we gently pipette water up and down (being careful not
to touch the meristem).
Acknowledgments
We acknowledge the Australian Research Council and European
Research Council for support.
References
1. Fernandez R, Das P, Mirabet V, Moscardi E,
Traas J, Verdeil JL, Malandain G, Godin C
(2010) Imaging plant growth in 4D: robust
tissue reconstruction and lineaging at cell
resolution. Nat Methods 7(7):547–553.
https://doi.org/10.1038/nmeth.1472
2. Bhatia N, Bozorg B, Larsson A, Ohno C,
Jonsson H, Heisler MG (2016) Auxin acts
Confocal Microscopy of Arabidopsis Seedlings
301