Correlative light and electron microscopy based on near-infrared branding as a tool for neurodevelopmental disorders research

Neurodevelopment relies on a tightly coordinated sequence of cellular events, precisely regulated across time and space. Genetic perturbations that disrupt this balance can lead to neurodevelopmental disorders: in this study, we focus on Angelmann Syndrome (AS) and Developmental and Epileptic Encephalopathy-9 (DEE9). Understanding how driver mutations exert context-dependent effects on cellular processes, and determining the relative contribution of distinct neuronal compartment to disease pathogenesis, is therefore crucial. Here, we describe how the access to the National Facility for Light Imaging at Human Technopole enabled the development and application of a volume correlative light and electron microscopy (vCLEM) pipeline for the volumetric ultrastructural analysis of targeted neurons. These investigations require refined genetic manipulation of animal models to selectively target specific neuronal populations, together with fluorescent labeling to identify cells exhibiting the phenotype of interest. However, when switching to electron microscopy, fluorescence is no longer a resource, posing a major challenge for accurate cell relocation across imaging modalities. To address this limitation, we leveraged the Zeiss LSM980 NLO two-photon microscope and implemented a near-infrared branding (NIRB) workflow. NIRB generates spatially confined fiducial marks using multiphoton excitation, which are essential for orienting the imaging setup when adopting serial-block face scanning electron microscopy. In close collaboration with the technical staff, we developed complementary coordinate-mapping strategies to efficiently target distinct neuronal compartments, including soma and dendrites, across excitatory and inhibitory neurons. This approach enables the ultrastructural investigation of genetically defined neurons within their native tissue context, providing an unprecedented view of synaptic organization in AS and DEE9, and revealing new layers of information that were previously inaccessible for this class of neurodevelopmental disorders. These insights are crucial for advancing fundamental knowledge, as well as for future translational research aimed at identifying novel therapeutic targets.