RNA-based CLEM (RCLEM) bridging RNA localisation and ultrastructural mapping in 3D

Area of Science:

• Molecular Biology
• Cell Biology
• Microscopy

Background:

• Spatial transcriptomics methods like single-cell RNA sequencing (scRNA-seq) map gene expression in tissues.
• Current spatial transcriptomics rely on light microscopy (LM), which has resolution limits for subcellular details.
• Existing correlative imaging methods struggle with RNA detection resolution due to LM optical constraints.

Purpose of the Study:

• To develop a high-resolution 3D workflow for visualizing RNA localization within the ultrastructural context of intact tissues.
• To overcome the resolution limitations of current light microscopy-based spatial transcriptomics techniques.
• To enable precise correlation between mRNA expression and cellular ultrastructure for advanced biological insights.

Main Methods:

• Developed a 3D RNA-based correlated light and electron microscopy (RCLEM) workflow.
• Integrated RNA labeling with serial block-face scanning electron microscopy (SBF-SEM).
• Optimized detergent conditions for probe accessibility in thick, whole-mount tissues while preserving ultrastructure.

Main Results:

• Achieved high-resolution 3D visualization of RNA molecules within their native ultrastructural environment.
• Demonstrated precise 3D correlation between mRNA expression and electron microscopy features in choroid plexus and liver tissues.
• Enabled detailed morphological characterization of rare cell subtypes defined by scRNA-seq.

Conclusions:

• The RCLEM workflow provides a powerful platform for integrative, high-resolution spatial transcriptomic analysis.
• This antibody-independent method bridges gene expression data with precise cellular morphology.
• The protocol is broadly applicable to diverse complex tissues for advanced spatial analyses.