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Dimeric gold nanoparticles enable multiplexed labeling in cryoelectron tomography.

Hoyoung Kim1, Cathy J Spangler1, Aya Matsui1,2

  • 1Vollum Institute, Oregon Health and Science University, Portland, OR 97239.

Proceedings of the National Academy of Sciences of the United States of America
|November 24, 2025
PubMed
Summary

Researchers developed dimeric gold nanoparticles (AuNPs) for multiplexed cryo-electron tomography (cryo-ET) imaging. This innovation allows distinguishing multiple molecular targets within cells, advancing nanoscale structural studies.

Keywords:
NMDA receptorscryoelectron tomographygold nanoparticlesmultiplexed labelingsynaptic cleft

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Area of Science:

  • Structural Biology
  • Nanotechnology
  • Biophysics

Background:

  • Cryoelectron tomography (cryo-ET) visualizes molecular structures in cells at nanometer resolution.
  • Fiducial labeling, especially with gold nanoparticles (AuNPs), is crucial for cryo-ET.
  • Multiplexing with conventional monomeric AuNPs is difficult, limiting multitarget studies.

Purpose of the Study:

  • To develop functionalized dimeric AuNPs for multiplexed labeling in cryo-ET.
  • To enable reliable discrimination between monomeric and dimeric AuNPs for identifying distinct molecular targets.
  • To validate the use of dimeric AuNPs for nanoscale molecular mapping in cellular environments.

Main Methods:

  • Synthesis of dimeric AuNPs with defined size (approx. 2.6 nm diameter) and spacing (approx. 1.4 nm).
  • Site-specific conjugation of anti-GluN1 Fab to dimeric AuNPs targeting N-methyl-D-aspartate receptors (NMDARs).
  • Application of a deep learning classifier for distinguishing monomeric and dimeric AuNPs in cryo-tomograms.

Main Results:

  • Structurally homogeneous dimeric AuNPs were validated by SAXS and electron microscopy.
  • Dimeric AuNP-Fab conjugates showed robust binding to recombinant NMDARs.
  • In situ cryo-ET confirmed dimeric labels reaching NMDARs in brain tissue synaptic clefts.

Conclusions:

  • Dimeric AuNPs provide a generalizable platform for distinguishable labels in cryo-ET.
  • This approach enables multiplexed labeling and identification of distinct molecular targets.
  • The compact and uniform dimeric AuNPs are suitable for nanoscale mapping in complex cellular environments.