In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in...

Area of Science:

• Plant cell biology
• Microscopy techniques
• Molecular imaging

Background:

• Correlative light and electron microscopy (CLEM) is crucial for nanometre-scale subcellular localization.
• Plant biology has faced challenges adopting CLEM due to tissue permeability and labeling issues.
• Existing methods struggle with indexing and re-localizing features in complex 3D plant tissues.

Purpose of the Study:

• To develop and validate an improved CLEM workflow for plant cells.
• To overcome limitations in plant tissue permeability and fluorescence labeling efficiency.
• To analyze DNA replication and nucleolar organization in Arabidopsis thaliana mutants.

Main Methods:

• Developed a CLEM workflow involving methacrylate resin embedding.
• Utilized single-molecule localization microscopy (SMLM) and transmission electron microscopy (TEM).
• Applied consecutive and same-section CLEM on ultrathin cross-sections of Arabidopsis thaliana.

Main Results:

• Methacrylate resin embedding is compatible with SMLM and improves fluorophore blinking.
• Successfully visualized DNA replication sites using Click-iT ethynyl-deoxyuridine labeling.
• Analyzed S-phase progression and nucleolar organization in wild-type and mutant Arabidopsis plants.

Conclusions:

• The developed in-section CLEM workflow is effective for plant cell imaging.
• This method facilitates the study of molecular processes and subcellular organization in plants.
• The approach is valuable for investigating plant mutants with aberrant nucleolar phenotypes.