Improved clearing method contributes to deep imaging of plant organs

Area of Science:

• Biological Imaging
• Microscopy Techniques
• Plant Science

Background:

• Tissue clearing is vital for observing thick biological samples.
• Previous methods like TOMEI had limitations in chlorophyll removal and fluorescence preservation.

Purpose of the Study:

• To develop an improved tissue clearing method (iTOMEI) that overcomes limitations of prior techniques.
• To enhance chlorophyll removal and preserve/restore fluorescence in biological tissues.

Main Methods:

• Developed iTOMEI using caprylyl sulfobetaine for chlorophyll removal.
• Incorporated a weak alkaline solution to restore GFP fluorescence.
• Utilized an iohexol solution for increased sample transparency.

Main Results:

• iTOMEI efficiently removed chlorophyll from Arabidopsis thaliana seedlings without quenching GFP.
• Restored GFP fluorescence lost during fixation and improved transparency.
• Achieved significantly brighter fluorescence detection in plant tissues (A. thaliana, Oryza sativa, Marchantia polymorpha) and mouse brains.

Conclusions:

• iTOMEI offers superior performance for tissue clearing compared to previous methods.
• The iTOMEI-Brain method provides efficient clearing and strong fluorescent signal detection in mammalian brains.
• This technique advances high-resolution imaging of internal biological structures.