Dual Color tau-STED Super Resolution Microscopy in Arabidopsis Root Tip

Area of Science:

• Plant Cell Biology
• Microscopy
• Biophysics

Background:

• Super-resolution microscopy offers nanoscale insights but faces challenges in complex plant tissues.
• Optical properties of plant tissues limit conventional high-resolution imaging.
• Visualizing fine subcellular structures in plants requires advanced techniques.

Purpose of the Study:

• To present a detailed protocol for tau-STED microscopy optimized for plant biology.
• To enable nanoscale visualization of subcellular structures in *Arabidopsis thaliana* root tips.
• To address specific challenges in plant tissue super-resolution imaging.

Main Methods:

• Utilized tau-STED microscopy, combining stimulated emission depletion (STED) and fluorescence lifetime imaging (FLIM).
• Employed time-correlated single-photon counting (TCSPC) for signal separation based on fluorescence lifetime.
• Developed a protocol covering sample prep, fluorophore choice, microscope setup, acquisition, and analysis.

Main Results:

• Achieved nanoscale resolution imaging in *Arabidopsis thaliana* root tips.
• Successfully visualized elusive subcellular compartments, including the endoplasmic reticulum-Golgi intermediate compartment (ERGIC).
• Demonstrated minimization of phototoxicity and addressed issues like autofluorescence and light scattering.

Conclusions:

• The tau-STED protocol provides a robust framework for high-detail plant cell biology research.
• This method is accessible to researchers with basic microscopy experience.
• Facilitates unprecedented exploration of subcellular dynamics in plants.