This method can be helpful in whole-plant imaging to reveal intact morphology, developmental processes and plant-microbe interactions. The main advantage of this technique is that we can directly observe the inside of a plant body without inflicting damage on it. As this method applies to a wide range of plant species and tissues, it can help to accelerate the discovery of new phenomena in plant biological research.
Fixation is a critical step in this procedure. Before the clearing step, one needs to check the intensity of fluorescent protein after fixation. To begin immerse plant samples in the fixative solution in a micro tube, and ensure that the volume of the fixative solution is more than five times the sample volume.
Seal the micro tube with a parafilm and make holes using a needle. Place the micro tube in a desiccator and slowly adjust the degree of vacuum to around 690 millimeters of mercury so that bubbles appear gradually from the samples. Turn off the vacuum pump after evacuating the desiccator.
Vent the desiccate carefully without disturbing the samples. Turn on the vacuum pump again and turn it off after evacuating the desiccator. Open the desiccator carefully without bumping the fixative solution in the microtube.
Remove the fixative solution and add 1x PBS with a micro pipette. After storing for one minute, replace the old PBS with new 1x PBS. After removing PBS at five times the sample volume of the clearing solution, seal the micro tube with parafilm and make holes with a needle.
Place the samples in the desiccator. Evacuate and turn off the vacuum pump. Open the desiccator gently, then close the microtube.
Invert the micro tube, every one to two days to accelerate the clearing process. For the spacer frame, cut a silicone rubber sheet with a razor blade adjusting the silicone rubber sheet thickness according to the sample thickness. Place the silicone frame on a cover glass, then place the treated samples within the frame and add around 100 microliters of clearing solution to remove any bubbles.
Cover with another cover glass to prevent evaporation of the clearing solution. Observe the samples under a fluorescent microscope. After observation return the samples to clearing solution in a microtube.
Fixed leaves of various species were incubated in PBS or ClearSee for eight days, in ClearSee or ClearSeeAlpha for two days. ClearSee can clear leaves of various species. After extracting the cuticular, ClearSee could clear rice leaves.
As the sodium sulfite component in ClearSeeAlpha prevents polyphenol oxidation due to the reducing effect, ClearSeeAlpha could clear tobacco and tirania pistils without any brown pigmentation. Ubiquitin-10 promoter H2B-mClover leaves of arabidopsis thaliana were treated with PBS or ClearSee for three days. ClearSee treatment reduced the pale green color of the arabidopsis H2B-mClover leaf and enhance the fluorescence intensity of H2B-mClover compared with PBS incubation.
ClearSee treated leaves were observed by two-photon excitation microscopy with 950 nanometer excitation. Cell wall is stained with calcaflour-white. Nuclei are labeled with ubiquitin-10 promoter, H2B-mClover.
The Y Z and X Z images are cross sections at the position indicated by the white dashed lines. Preparation of the fixative solution is important to fix the samples. However, care should be taken to prevent damage to the samples under vacuum.
Microscopic imaging can be performed to study multiple gene expression patterns and intracellular communications during plant development and infection following this procedure.
