Materials and Reagents

>100 ml container to hold the staining solution (see procedure A) during rotational incubation
An incubation container for staining (e.g., 1.5 ml micro tube, Petri dish)
Super frost slide glass (Matsunami Glass Ind., catalog number: S2441 )
NEO microscope coverslip, 24 x 32 mm (Matsunami Glass, catalog number: C024321 )
Parafilm M^® (Bemis Company, Inc., catalog number: PM-996 )
Pipetman p200 or 1 ml disposable transfer pipette
Silicone oil (Shin-Etsu Chemical Co., catalog number: KF96L-1.0 cs or KF96L-1.5 cs )
Note: It is named “Dimethyl Silicone Fluid” on Shin-Etsu Chemical Co. website.
Nile Red (Invitrogen, catalog number: N-1142 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: N-1142”.

Equipment

Tube rotator or shaker
Electronic scale
Confocal microscope (Carl Zeiss Microscopy, model: LSM-780) or fluorescence microscope (Carl Zeiss Microscopy, model: Axioskop2 plus)
Sonication bath (HONDA ELECTRONICS Co., model: W-113 )
Note: This is not absolutely necessary.

Procedure

Preparation of staining solution
1. Add 0.318 mg of Nile Red and 100 ml of silicone oil (KF96L-1.0 cs or KF96L-1.5 cs) to a container. The final concentration of this solution is 10 μM. About 100 samples can be prepared from this amount of staining solution if you use 1.5 ml microtubes for staining.
2. Tightly seal the container with Parafilm M^®.
3. (Optional) Sonicate the suspended Nile Red using a sonication bath (10 min, 100 kHz) to make Nile Red dissolved faster.
4. Wrap the container with aluminum foil, place the tube on a tube rotator and rotate for 1-2 d.
5. (Optional) Undissolved dye can be removed via filtration or centrifugation.
Staining
1. Remove a small (5 x 5 ~ 10 x 10 mm) section of the leaf tissue to be examined.
  Note: When you conduct time-lapse imaging, skip this step and stain a whole plant.
2. Add the specimen and staining solution to an incubation container.
3. Incubate specimen in staining solution for 5 min (adaxial side) or 15 min (abaxial side).
Slide preparation
Prepare slides according to Figure 1.

Figure 1. Procedure of slide preparation
Microscopy
The steps B-C fill airspaces with Nile Red in silicone oil. Image airspaces (excitation wavelength 488, emission wavelength 490-560) and cells (excitation wavelength 561, emission wavelength 563-639). Representative data is shown in Figure 2.
(Optional) Procedures for time-lapse imaging
After observation, shake the plants in silicone oil (KF96L-1.0 cs) gently at 30~60 rpm for 1 min twice to remove Nile Red. Put the plants on culture beds until the next observation. Repeat the steps B-E. Representative data of time-lapse imaging is shown in Figure 3.
Image segmentation
You can extract shapes of cells and airspaces from acquired 3D images by using DSLT_Demo. Step by step instructions for segmentation are available at http://dslt.bot.kyoto-u.ac.jp/DSLT_manual_v111.pdf.

Representative data

Figure 2. Visualizing cells and airspaces in an Arabidopsis thaliana leaf. Confocal images of the epidermis (left) and mesophyll (right) of an Arabidopsis thaliana leaf. The abaxial side of the leaf was stained with solution of Nile Red in silicone oil and imaged with 488 nm excitation for Nile Red in silicone oil (airspace, green) and 561 nm excitation for Nile Red in lipid (cellular membrane, magenta). Asterisks indicate airspaces. Scale bars: 50 μm.

Figure 3. Time-lapse imaging of a developing Nicotiana glauca leaf. Cross-sectional views of 3D images of the adaxial side of a Nicotiana glauca leaf. Confocal images were taken over 5 days of leaf development (times indicated in white above each image). Shown is the fluorescence of Nile Red in the lipid bilayer membrane. Bar is 50 μm.

Acknowledgments

This protocol was adapted from the research article Kawase et al. (2015). The work was supported by a Specially Promoted Research of Grant-in-Aid for Scientific Research to I. H. -N. (no.22000014) from the Japan Society for the Promotion of Science (JSPS).

References

Kawase, T., Sugano, S. S., Shimada, T. and Hara-Nishimura, I. (2015). A direction-selective local-thresholding method, DSLT, in combination with a dye-based method for automated three-dimensional segmentation of cells and airspaces in developing leaves. Plant J 81(2): 357-366.