Expansion microscopy of banked brain tissue.

Expansion microscopy (ExM) physically enlarges biological specimens to enable ultrastructural imaging with conventional fluorescence microscopes. However, its performance in postmortem human brain tissue is unclear. Here, we evaluated how a previously established ExM protocol performs on cortical tissue from eight banked brains with postmortem intervals ranging from 40 minutes to 91 hours, and compared these data with electron microscopy (EM) on a subset of matched samples. Both techniques revealed similar patterns of ultrastructural features, including irregular, rounded, unstained spaces as postmortem artifacts that increased with longer postmortem intervals. While EM provided superior resolution for synaptic details, ExM enabled more high-throughput volumetric imaging of neural circuits. ExM also enabled molecular annotation of ultrastructure through immunofluorescence, as demonstrated by SMI-312 neurofilament and γ-protocadherin labeling. Our findings show that ExM can visualize aspects of ultrastructure in routinely banked brain tissue. While EM provides better resolution for fine synaptic detail, ExM offers a complementary approach that combines nanoscale imaging with molecular specificity and more accessible high-throughput volumetric capabilities.