2. Academic Validation
  3. Dense, continuous membrane labeling and expansion microscopy visualization of ultrastructure in tissues

Dense, continuous membrane labeling and expansion microscopy visualization of ultrastructure in tissues

  • Nat Commun. 2025 Feb 12;16(1):1579. doi: 10.1038/s41467-025-56641-z.
Tay Won Shin 1 2 3,  Hao Wang # 1 4,  Chi Zhang # 1,  Bobae An 1,  Yangning Lu 1,  Elizabeth Zhang 1,  Xiaotang Lu 5,  Emmanouil D Karagiannis 1,  Jeong Seuk Kang 1,  Amauche Emenari 1 2,  Panagiotis Symvoulidis 1,  Shoh Asano 1 6,  Leanne Lin 7,  Emma K Costa 2 8,  IMAXT Grand Challenge Consortium,  Adam H Marblestone 1 9,  Narayanan Kasthuri 10 11,  Li-Huei Tsai 2 4,  Edward S Boyden 12 13 14 15 16 17 18 19
Affiliations

Affiliations

  • 1 McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
  • 2 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
  • 3 Department of Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02140, USA.
  • 4 Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
  • 5 Department of Cellular and Molecular Biology, Harvard University, Cambridge, MA, 02138, USA.
  • 6 Pfizer Inc, Cambridge, MA, 02139, USA.
  • 7 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
  • 8 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford University, Stanford, CA, 94305, USA.
  • 9 Convergent Research, Cambridge, MA, 02140, USA.
  • 10 Center for Nanoscale Materials, Argonne National Laboratory, Lemont, IL, 60439, USA.
  • 11 Department of Neurobiology, University of Chicago, Chicago, IL, 60637, USA.
  • 12 McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. [email protected].
  • 13 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. [email protected].
  • 14 Department of Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02140, USA. [email protected].
  • 15 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. [email protected].
  • 16 Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. [email protected].
  • 17 Center for Neurobiological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. [email protected].
  • 18 K. Lisa Yang Center for Bionics, Cambridge, MA, 02139, USA. [email protected].
  • 19 Howard Hughes Medical Institute, Cambridge, MA, 02139, USA. [email protected].
  • # Contributed equally.
PMID: 39939319 DOI: 10.1038/s41467-025-56641-z
Abstract

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines an innovative membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validate the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supports the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrate the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yields ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

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