PT  - JOURNAL ARTICLE
AU  - Ons M’Saad
AU  - Ravikiran Kasula
AU  - Ilona Kondratiuk
AU  - Phylicia Kidd
AU  - Hanieh Falahati
AU  - Juliana E. Gentile
AU  - Robert F. Niescier
AU  - Katherine Watters
AU  - Robert C. Sterner
AU  - Seong Lee
AU  - Xinran Liu
AU  - Pietro De Camilli
AU  - James E. Rothman
AU  - Anthony J. Koleske
AU  - Thomas Biederer
AU  - Joerg Bewersdorf
TI  - All-optical visualization of specific molecules in the ultrastructural context of brain tissue
AID  - 10.1101/2022.04.04.486901
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.04.04.486901
4099  - http://biorxiv.org/content/early/2022/04/17/2022.04.04.486901.short
4100  - http://biorxiv.org/content/early/2022/04/17/2022.04.04.486901.full
AB  - 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.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studiesCompeting Interest StatementJ.B. has financial interests in Bruker Corp. and Hamamatsu Photonics. O.M., J.E.R. and J.B. filed patent applications with the U.S. patent office covering the presented method. O.M. and J.B. are co-founders of panluminate Inc. which is developing related products.