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

Spatial transcriptomics and in situ hybridisation techniques have become essential for contextualising single-cell RNA sequencing (scRNA-seq) data by mapping cell type- and state-specific mRNA expression within intact tissues. Beyond validating scRNA-seq predictions, these spatial methods provide critical insight into the localisation of specific cell types and their spatial relationships with neighbouring cells. However, current approaches rely on light microscopy (LM), which, despite advances, lacks the resolution needed to resolve detailed cell morphology or subcellular RNA localisation in situ. While correlative imaging methods can enhance spatial context, RNA detection remains limited by the optical constraints of LM. To overcome these challenges, we developed a three-dimensional RNA-based correlated light and electron microscopy (RCLEM) workflow. This method integrates RNA labelling with serial block-face scanning electron microscopy (SBF-SEM), allowing high-resolution 3D visualisation of RNA molecules within their ultrastructural environment. We optimised the RNA labelling protocol for thick, whole-mount tissues by fine-tuning detergent conditions to preserve ultrastructure while maintaining probe accessibility. This RCLEM approach offers several advantages over protein-based correlative methods, including robust detection with nucleotide probes and independence from antibody optimisation or fixation-sensitive epitopes. Applied to choroid plexus (ChP) and liver tissue, this workflow enabled precise 3D correlation between mRNA expression and EM features, supporting detailed morphological characterisation of rare scRNA-seq-defined cell subtypes. This protocol is broadly applicable to other complex tissues and provides a powerful platform for integrative, high-resolution spatial transcriptomic analyses that bridge gene expression with ultrastructure. LAY DESCRIPTION: To overcome the resolution limits of current spatial transcriptomics, we developed a 3D RNA-based correlated light and electron microscopy (RCLEM) workflow. By integrating RNA labelling with scanning electron microscopy, this method enables high-resolution 3D visualisation of mRNA within its ultrastructural environment in thick tissues. Validated in choroid plexus and liver, our antibody-independent RCLEM approach provides a broadly applicable platform bridging gene expression and precise cellular morphology for advanced spatial analyses.