Deep Fluorescence Observation in Rice Shoots via Clearing Technology

Area of Science:

• Plant biology
• Developmental biology
• Microscopy

Background:

• Clearing technology enables 3D visualization of plant tissues by reducing refractive index mismatches and auto-fluorescence.
• Previous clearing methods had limited success in rice (Oryza sativa L.) shoot apical meristems (SAM) and stems due to poor clearing solution penetration.
• Tissue characteristics like auto-fluorescence, thickening, and water-repellent layers impede clearing in these specific rice organs.

Purpose of the Study:

• To optimize a clearing approach for continuous, 3D observation of gene expression in rice SAM and stems.
• To overcome limitations in clearing solution penetration in difficult-to-clear rice tissues.
• To enable macro-level observation of internal shoot structures from SAM to the base.

Main Methods:

• Developed an optimized clearing protocol for rice (Oryza sativa L.) shoot tissues.
• Utilized a vibrating micro-slicer to trim fixed, fluorescent protein-expressing tissue samples to an appropriate thickness.
• Applied clearing solution (CS) with targeted application to the central tissue to enhance penetration and uniformity.

Main Results:

• Achieved increased penetration rate and uniformity of the clearing solution in rice shoot tissues.
• Significantly reduced the time required to render the tissues transparent.
• Enabled macro-scale observation of the internal structure of the entire shoot from SAM to the base.

Conclusions:

• The optimized clearing method facilitates deep imaging and 3D observation of gene expression in challenging rice (Oryza sativa L.) shoot tissues.
• This protocol enhances visualization of internal plant structures, overcoming previous limitations in SAM and stem clearing.
• The technique shows potential for application in clearing other difficult-to-image plant species.