A Correlative Light and Electron Microscopy Workflow for Nanoscale Three‐Dimensional Reconstruction of Synaptic Projections From the Thalamic Reticular Nucleus

Correlative light and electron microscopy (CLEM) combines molecular specificity with nanoscale ultrastructural resolution and has become an important approach for investigating neural circuit organization. However, ultrastructural reconstruction of long-range projection terminals remains technically challenging. This study aimed to develop a CLEM workflow for the identification, localization, three-dimensional (3D) reconstruction, and ultrastructural characterization of thalamic reticular nucleus (TRN)-derived presynaptic terminals in the mouse thalamus. TRN presynaptic terminals were selectively labeled by stereotaxic injection of rAAV-hSyn-Synaptophysin-mCherry. Confocal microscopy was combined with serial section scanning electron microscopy (ssSEM) to achieve fluorescence-guided ultrastructural imaging and 3D reconstruction of labeled axonal boutons and synaptic contacts. The CLEM workflow enabled fluorescence-guided identification of presynaptic terminals within serial EM datasets. 3D reconstruction of a representative TRN axon segment revealed multiple en passant boutons along continuous axonal shafts. Ultrastructural analysis demonstrated synaptic contacts with multiple postsynaptic dendritic profiles, together with distinct vesicle distributions and mitochondrial organization within presynaptic boutons. This study presents a practical CLEM-based workflow for nanoscale reconstruction of long-range synaptic projections. Application of this method to the TRN-to-thalamus pathway illustrates the spatial organization of presynaptic boutons and their postsynaptic contacts in the thalamic neuropil. The workflow provides a methodological framework for ultrastructural studies of defined neural projections in the mammalian brain.