Abstract
OBJECTIVE: Transmission electron microscopy (TEM) enables ultrastructural investigation of both organic and nonorganic samples. However, conventional TEM is limited by the acquisition of two-dimensional snapshots, restricting our volumetric understanding of complex ultrastructures. Electron tomography (ET) overcomes this limitation by offering detailed three-dimensional (3D) specimen representation. ET has been widely applied in biology; however, its use for blood-brain barrier (BBB) assessment has been overlooked. The BBB ensures proper brain function by limiting the entrance of blood-borne molecules into the brain and ensuring selective transport. The BBB is disrupted in several pathological conditions, resulting in neuronal damage. Understanding the fine changes underlying BBB disruption requires advanced imaging tools such as ET. METHODS: We developed a detailed room temperature electron tomography (RT-ET) method for sample preparation, tomogram generation, 3D segmentation, and applied this approach to assess ultrastructural changes in brain endothelial cells (ECs) after photothrombotic stroke in mice. RESULTS: Our findings identify altered transcytotic vesicle morphology, as well as remodeling of the endoplasmic reticulum, indicative of cellular stress and impaired vesicular trafficking. CONCLUSIONS: Our toolkit allows for reproducible, high-resolution analysis of brain microvascular pathology. This new RT-ET approach uncovers previously inaccessible ultrastructural alterations in ECs following ischemic stroke in mice, offering new insight into mechanisms of BBB disruption.