protein can be useful depending on which cell types are being
targeted for analysis. However, and as mentioned above, the
expression of the LacI-GFP should not be very strong as the
background fluorescence from unbound LacI-GFP may mask
the signal coming from the targeted locus.
2. Depending on the experimental setup, day-night periods, temperature, and light intensity could be changed according to the
goal of the study, but these should be kept constant during
experimental replicates.
3. Growing plants vertically allows roots to grow straight along
the surface of the media, which facilitate the transfer of the
seedlings to the microscope slide and avoid damaging the
samples.
4. Mounting the roots only between a microscope slide and a
cover slip without tape is possible but it increases the possibility
to squash and damage the root. Adding a double-sticky tape
creates a spacer, therefore avoiding squashing the roots. Additionally, it seals the slide and avoids drying of the sample during
imaging. We advise this specific tape type (Grace Bio-Labs)
because of its size and efficiency in sealing, as other tapes
often go off in contact with water.
5. Laser scanning confocal microscopes can be limiting for 3D
imaging of very dynamic processes due to a slow scan speed. A
trade-off between resolution (i.e., image quality and the number of z-steps) and scanning speed is necessary to allow
dynamic observations. Spinning disc confocal microscopes provide faster-imaging systems and therefore are likely to be advantageous for quantifying highly dynamic motions.
6. Using a high magnification, such as 63Â, allows collecting
5–10 nuclei at the same time with good resolution, which is
important for a proper nucleus alignment and spot tracking.
7. After 1 week of germination, Arabidopsis seedlings are long
enough for imaging, but the time can be adjusted depending
on the experimental purpose.
8. In order to limit movement of the cell imaged along the x–y
axis, the seedling is immobilized by placing the upper part of
the root between the double-sticky tape and the coverslip while
the cotyledons are left outside the chamber (as shown in
Fig. 1). Nevertheless and even though the imaging time is
relatively short (approx. 5 min) the nuclei may still move along
the x–y axis due to cell expansion. It is therefore necessary to
limit this movement and to ensure, post-imaging, the correct
alignment of the nucleus between the different frames.
Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana
221
targeted for analysis. However, and as mentioned above, the
expression of the LacI-GFP should not be very strong as the
background fluorescence from unbound LacI-GFP may mask
the signal coming from the targeted locus.
2. Depending on the experimental setup, day-night periods, temperature, and light intensity could be changed according to the
goal of the study, but these should be kept constant during
experimental replicates.
3. Growing plants vertically allows roots to grow straight along
the surface of the media, which facilitate the transfer of the
seedlings to the microscope slide and avoid damaging the
samples.
4. Mounting the roots only between a microscope slide and a
cover slip without tape is possible but it increases the possibility
to squash and damage the root. Adding a double-sticky tape
creates a spacer, therefore avoiding squashing the roots. Additionally, it seals the slide and avoids drying of the sample during
imaging. We advise this specific tape type (Grace Bio-Labs)
because of its size and efficiency in sealing, as other tapes
often go off in contact with water.
5. Laser scanning confocal microscopes can be limiting for 3D
imaging of very dynamic processes due to a slow scan speed. A
trade-off between resolution (i.e., image quality and the number of z-steps) and scanning speed is necessary to allow
dynamic observations. Spinning disc confocal microscopes provide faster-imaging systems and therefore are likely to be advantageous for quantifying highly dynamic motions.
6. Using a high magnification, such as 63Â, allows collecting
5–10 nuclei at the same time with good resolution, which is
important for a proper nucleus alignment and spot tracking.
7. After 1 week of germination, Arabidopsis seedlings are long
enough for imaging, but the time can be adjusted depending
on the experimental purpose.
8. In order to limit movement of the cell imaged along the x–y
axis, the seedling is immobilized by placing the upper part of
the root between the double-sticky tape and the coverslip while
the cotyledons are left outside the chamber (as shown in
Fig. 1). Nevertheless and even though the imaging time is
relatively short (approx. 5 min) the nuclei may still move along
the x–y axis due to cell expansion. It is therefore necessary to
limit this movement and to ensure, post-imaging, the correct
alignment of the nucleus between the different frames.
Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana
221