ClearSeeAlpha: Advanced Optical Clearing for Whole-Plant Imaging

Area of Science:

• Plant biology
• Microscopy
• Biotechnology

Background:

• Plant internal structures are difficult to observe due to opacity.
• Existing clearing reagents like ClearSee have limitations with certain plant species and tissues.
• Direct observation of intact plant tissues is crucial for understanding morphology and cellular arrangement.

Purpose of the Study:

• To improve plant tissue transparency for microscopic observation.
• To develop a clearing reagent effective across a broader range of plant species and tissues.
• To address limitations of previous clearing methods, specifically oxidation-induced browning.

Main Methods:

• Modification of the ClearSee reagent composition to include sodium sulfite, a reductant.
• Testing the efficacy of the new reagent (ClearSeeAlpha) on various plant species and tissues, including chrysanthemum, tobacco, Torenia, and Arabidopsis thaliana.
• Assessing the impact of ClearSeeAlpha on the fluorescence intensity of various fluorescent proteins.

Main Results:

• ClearSeeAlpha successfully rendered previously opaque plant tissues transparent, including chrysanthemum leaves, tobacco and Torenia pistils, and fertilized Arabidopsis thaliana fruits.
• The addition of sodium sulfite prevented brown pigmentation caused by oxidation during the clearing process.
• Fluorescence intensity of most tested fluorescent proteins remained unaffected, with only TagRFP showing a partial reduction.

Conclusions:

• ClearSeeAlpha offers enhanced transparency for a wider array of plant samples compared to ClearSee.
• The improved reagent facilitates detailed observation of internal plant structures without physical sectioning.
• ClearSeeAlpha is a valuable tool for advancing research in plant biology by enabling the study of deep tissue phenomena.