Kavli Affiliate: Joerg Bewersdorf, Pietro DeCamilli
| Authors: Ons M’Saad, Ravikiran Kasula, Ilona Kondratiuk, Phylicia Kidd, Hanieh Falahati, Juliana E Gentile, Robert F Niescier, Katherine Watters, Robert C Sterner, Seong Lee, Xinran Liu, Pietro De Camilli, James E Rothman, Anthony J Koleske, Thomas Biederer and Joerg Bewersdorf
| Summary:
Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community. Highlights pan-ExM-t visualizes proteins in the context of synaptic ultrastructure Lipid labeling in pan-ExM-t reveals organellar and cellular membranes All-optical, easily accessible alternative to correlative light/electron microscopy High potential for high throughput connectomics studies