Plant Optical Clearing Reagent

ClearSee™

Dr. Daisuke Kurihara (Nagoya University) developed the clearing solution, termed ClearSee™, to diminish chlorophyll auto fluorescence while maintaining fluorescent protein stability and tissue structures of plant samples. ClearSee™ is applicable to multi colors deep imaging of various plant samples without sectioning.




Image data courtesy of:Daisuke Kurihara and Yoko Mizuta from Graduate School of Science, Nagoya University, Nagoya, Japan

Features

  • Procedures are only 3 steps
  • Every fluorescence microscopes can be used
  • ClearSee™-treated samples can be store for long term (at least 6 months)
  • The ClearSee™-treated samples can be observed for many times

Protocol

Fixative solution

  • Add 8 mL of sterilized water in a conical tube.
  • Add 0.4 g of paraformaldehyde (in the draft chamber).
  • After adding 50 µL of 2mol/L Sodium Hydroxide Solution, close and seal a tube with parafilm (in the draft chamber).
  • Incubate at 60°C until clearing with occasionally inversion.
  • After dissolving and cooling, add 1 mL of 10x PBS to adjust pH (in the draft chamber).
  • Adjust 10 mL by sterilized water (in the draft chamber).
    (Preparation freshly is better, but fixative solution can be stored at -30°C.)

Fixation

1. Put plant samples in a microtube and add 1.3 mL of fixative solution (in the draft chamber).

[Notes]
The sample volume should not exceed 20% of the volume of fixative solution. If the sample volume is larger, prepare large centrifuge tube and increase fixative solution. Because the samples are floated, soak the samples into fixative solution as possible.

Fig.1 Arabidopsis leaves and seedling in fixative solution

2. To prevent the escape of samples, seal the microtube with paraffin film and open holes by a needle.

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Fig.2 Open holes on the parafilm

3. Put the samples in the desiccator and turn on the vacuum pump. After closing a desiccator and turn off the vacuum pump, fix for 30 minutes at room temperature.

[Notes]
Vacuum degree depends on plant species and tissue type (eg. -90 kPa for Arabidopsis). Strong vacuum induces the damage of the samples. Adjust vacuum degree to appear slowly bubbles from the samples.

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Fig.3 Bubbles from the samples in a microtube

4. Slowly open the desiccator and turn on again the vacuum pump. After closing a desiccator and turn off the vacuum pump, fix for 30 minutes at room temperature.

[Notes]
Take care to prevent the damage of samples by open the desiccator. Two times of vacuum treatment enhance the penetration of fixative solution into the samples. When fixative solution penetrates into the samples, the samples are soaked after brief centrifugation (Fig.4). If the samples are floated even after centrifugation, additional vacuum treatment are required.

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Fig.4 Arabidopsis leaves and seedling after fixative treatmenent

Wash

5. Slowly open the desiccator. After removing the fixative solution, add 1 mL of 1 x PBS and store for 1 minute (in the draft chamber).
6. After removing 1 x PBS, add new 1 mL of 1 x PBS and store for 1 minute.

Cleaning

7. After removing 1 x PBS, add 1.3 mL of ClearSee™.
8. Seal the microtube with parafilm and open holes by a needle. Put the samples in the desiccator and turn on the vacuum pump. After closing a desiccator and turn off the vacuum pump, store for 1 hour at room temperature.

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Fig. 5 Arabidopsis leaves and seedling in ClearSee™ solution

9. Slowly open the desiccator. After removing the parafilm and close the microtube, store at room temperature in the dark for clearing.

[Notes]
Invert the microtube every 1-2 days to diffuse the chlorophyll. Required incubation times depends on plant species and tissue type. For examples, the incubation times for clearing are 1~2 days for roots, 4~7 days for leaves and seedlings, 2 weeks for pistils, and 4 weeks for mature tissues.

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Fig. 6 Chlorophyll are observed around the samples (1~2 days incubation)

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Fig. 7 Invert the tube

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Fig. 8 Substitute new ClearSee™

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Fig. 9 Cleared Arabidopsis leaves and seedling (3~5 days incubation)

Observation

10. Put vaseline on a slide glass.
11. Transfer the ClearSee™ -treated samples into ClearSee™ on a slide glass.
12. Cover with a cover glass and seal with the cover glass with vaseline.
13. Observe the ClearSee™ -treated samples under a fluorescent microscope.

[Notes]
Take care to prevent the damage of samples by transfer the samples. To prevent the evaporation of ClearSee™ by vaseline because of deposition. The samples can be stored into ClearSee™ in a microtube after observation.

[Points]
Fixation step is important for clearing without the damage of samples. To investigate the fixation conditions of plant samples for the first time, observe the fluorescent proteins in the samples after fixation.

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Fig. 10 The ClearSee™-treated samples are cleared under a bright field. Autofluorescence of chlorophyll are diminished. The fluorescent proteins are detected even in the vascular bundles. Scale bar : 200 µm.

Image1

Image of ClearSee™ -treated samples

ClearSee™ is applicable to various plant species other than Arabidopsis. Fig. 11 shows ClearSee™-treated leaves of torenia, petunia, tobacco, tomato, cucumber, and rice after 6-days incubation. In the case of rice, removal of cuticular wax are required for penetration of solution. Immerse the rice samples into organic solvents such as chloroform for 10~30 seconds and then fix with a fixative solution.

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Fig. 11 ClearSee™-treated leaves of various plant species (6-days incubation)

Image2

Image of ClearSee™ for multi color imaging of the whole pistil

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Left: a whole pistil before ClearSee™ process
Right: a whole pistil after ClearSee™ process

Image3

Image of ClearSee™ -treated Arabidopsis root

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Upper: a Arabidopsis root before ClearSee™ process
Bottom: a Arabidopsis root after ClearSee™ process

References

1) Kurihara, D. et al. : Development, 142, 4168-4179 (2015).
2) Nagaki ,K. et al. : Scientific Reports, 7, 42203(2017).
3) Ohtsu,M. et al. : Protoplasma (2017).
4) Kalmbach,L. et al.: Nature plants, 3,17058(2017).

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