2. The PDMS perfusion chamber is sterilized by autoclaving
(details 121
C for 15 min) before use and can be reused by
washing with ultrapure water and autoclaving.
3. We obtained the holder for the aluminum perfusion chamber
to fit our microplate stage from a local metal company
(Fig. 3b). Design of the chamber holder depends upon your
microscope stage. You can also make a resin-based holder using
a 3D-printer.
4. Confocal laser scanning microscopy (CLSM) enables higher
signal-to-noise ratio imaging with higher spatial resolution
than epifluorescence microscopy. However, because CLSM
takes longer to acquire images due to the scanning step, temporal resolution of CLSM is generally lower than epifluorescence microscopy.
5. Fiji is the ImageJ-based image processing software with numerous plugins which are useful for scientific image analysis.
6. The plant growth medium should be modified according to
your experiment. When sucrose-free medium is used for germination, some plant genotypes show a reduction in primary
root elongation. In this case, you can change to a medium with
1% (w/v) sucrose.
7. To prevent drying of the medium in the plastic cones, do not
prepare and store them for days. We make the plastic cones
right before sowing seeds.
8. Sterilization solution can be substituted to 1% (v/v) hypochlorous acid with 0.04% (v/v) Triton X-100.
9. Timing of root exit from the pipette tip depends on the genotype and conditions. You can modify the timing when you
observe the roots.
10. Optimal excitation wavelength paths are 485 and 437 nm for
cpsfGFP and LSSmOrange, respectively. There are multiple
options—either a compromise or separate excitation wavelengths at the optimal for each FP. In this chapter, we used
470 nm and 440 nm for excitation of cpsfGFP and LSSmOrange, respectively.
11. Exposure time can be changed according to the fluorescence
intensity. However, exposure time that are too long cause
photobleaching of the FPs and low temporal resolution of
the images.
12. The capture interval should be adjusted since too much image
acquisition causes photo-bleaching of FPs and produces too
much imaging data, requiring more storage infrastructure.
318
Akira Yoshinari et al.
(details 121
C for 15 min) before use and can be reused by
washing with ultrapure water and autoclaving.
3. We obtained the holder for the aluminum perfusion chamber
to fit our microplate stage from a local metal company
(Fig. 3b). Design of the chamber holder depends upon your
microscope stage. You can also make a resin-based holder using
a 3D-printer.
4. Confocal laser scanning microscopy (CLSM) enables higher
signal-to-noise ratio imaging with higher spatial resolution
than epifluorescence microscopy. However, because CLSM
takes longer to acquire images due to the scanning step, temporal resolution of CLSM is generally lower than epifluorescence microscopy.
5. Fiji is the ImageJ-based image processing software with numerous plugins which are useful for scientific image analysis.
6. The plant growth medium should be modified according to
your experiment. When sucrose-free medium is used for germination, some plant genotypes show a reduction in primary
root elongation. In this case, you can change to a medium with
1% (w/v) sucrose.
7. To prevent drying of the medium in the plastic cones, do not
prepare and store them for days. We make the plastic cones
right before sowing seeds.
8. Sterilization solution can be substituted to 1% (v/v) hypochlorous acid with 0.04% (v/v) Triton X-100.
9. Timing of root exit from the pipette tip depends on the genotype and conditions. You can modify the timing when you
observe the roots.
10. Optimal excitation wavelength paths are 485 and 437 nm for
cpsfGFP and LSSmOrange, respectively. There are multiple
options—either a compromise or separate excitation wavelengths at the optimal for each FP. In this chapter, we used
470 nm and 440 nm for excitation of cpsfGFP and LSSmOrange, respectively.
11. Exposure time can be changed according to the fluorescence
intensity. However, exposure time that are too long cause
photobleaching of the FPs and low temporal resolution of
the images.
12. The capture interval should be adjusted since too much image
acquisition causes photo-bleaching of FPs and produces too
much imaging data, requiring more storage infrastructure.
318
Akira Yoshinari et al.