vCLEM: bridging the gap between high-resolution structural imaging and functional analysis of synapses

Understanding the sophisticated details of synaptic structures is crucial when studying the mechanisms of neuronal communication and brain organization in health and disease. Traditional imaging techniques often fail to meet the necessity of providing both the detailed ultrastructure and the specific protein localization we need for comprehensive synaptic studies. Correlative Light and Electron Microscopy (CLEM) addresses this challenge by imaging the same exact biological structure with two, or more, imaging modalities, thus combining the strengths of light with the resolution of electron microscopy. Volume CLEM (vCLEM) adds a higher level of complexity to this already intricated method, introducing the invaluable benefits of the third dimension. We use vCLEM to study Angelman Syndrome (AS), a neurogenic disorder resulting from the loss of the maternal copy of the UBE3A gene. Taking advantage of CRISPR/Cas9- mediated genome editing endogenous Ube3a is inactivated via sparse in utero electroporation (IUE) of layer 2/3 pyramidal neurons of the mouse somatosensory cortex. The correlative workflow is complex and involves several steps: first, a confocal microscope is used to identify electroporated fluorescent neurons in the brain section and acquire images; next, Near Infra-Red Banding (NIRB) is conducted to generate spatial coordinates for locating the electroporated neurons along the x, y, and z axes; the sample is processed for the Serial Block-Face Scanning Electron Microscope (SBF- SEM), which is then exploited to create volumetric datasets. This approach represents a significant advancement in the study of synapses and allows us to gain a comprehensive understanding of the effect of the loss of Ube3a gene on synapse ultrastructure and brain connectivity during development.