Biological Sciences Division
Published February 12, 2025 | Version v1
Journal article Open

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

Creators

  • 1. Massachusetts Institute of Technology
  • 2. Harvard University
  • 3. Pfizer Inc
  • 4. Stanford University
  • 5. Convergent Research
  • 6. University of Chicago

Description

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.

Data availability

The raw and processed image stack data generated with umExM in this study are available on the Open Science Framework at https://osf.io/qtbek/. Source data are provided with this paper.

The source code for analyzing umExM data is available on GitHub at https://github.com/TAYmit/umExM

Files

Dense-continuous-membrane-labeling-and-expansion-microscopy-visualization-of-ultrastructure-in-tissues.pdf

Files (294.7 MB)

Additional details

Identifiers

DOI
10.1038/s41467-025-56641-z
Other
oai:uchicago.tind.io:14553

Funding

Lisa Yang
Y. Eva Tan
John Doerr
Open Philanthropy Project
MIT Media Lab
HHMI
U.S. Army Research Laboratory
U.S. Army Research Office
W911NF1510548
Cancer Research UK Grand Challenge
C9545/A24042
Brain and Cognitive Sciences Department, MIT
New York Stem Cell Foundation
Robertson Investigator Award
National Institutes of Health
Transformative Award
National Institutes of Health
Director’s Pioneer Award
National Institutes of Health
1R01EY023173
National Institutes of Health
1U01MH106011
MIT
McGovern Institute MINT program
Lore McGovern
Tom Stocky
Avni Shah
Kathleen Octavio
Good Ventures
National Institutes of Health
1R01AG070831
National Institutes of Health
1R01MH123403
National Institutes of Health
R01MH124606
National Institutes of Health
R01AG087374
Schmidt Futures
Nationall Science Foundation
Graduate Research Fellowship
Unknown funder
J. Douglas Tan Postdoctoral Fellow for Autism Research
Samsung Scholarship

UChicago Information

Division(s)
Biological Sciences Division
Department(s)
Neurobiology