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Multiplexed Single-Particle Imaging Enabled by Modulation of Er3+ Energy-Level Populations.

Wenrui Zhang1, Fei Du1, Tianli Zhai1

  • 1Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Fudan University, Shanghai, 200438, China.

Advanced Materials (Deerfield Beach, Fla.)
|September 20, 2025
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Summary

This study introduces a new non-photobleaching imaging method using upconversion nanoparticles (UCNPs) for long-term multicolor single-particle tracking (SPT). This technique enables multiplexed tracking of molecular dynamics in live cells with high precision.

Keywords:
energy transferlanthanidemultiplexingreceptor‐mediated endocytosissingle‐particle tracking

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Area of Science:

  • Nanotechnology and Materials Science
  • Biophysics and Molecular Imaging

Background:

  • Single-particle tracking (SPT) is crucial for studying molecular dynamics but is limited by photobleaching and complex multicolor imaging.
  • Existing methods face challenges with short tracking durations and technical hurdles in distinguishing multiple simultaneously tracked particles.

Purpose of the Study:

  • To develop a non-photobleaching, ratiometric imaging strategy for multiplexed single-particle tracking (SPT).
  • To enable long-term, multicolor imaging of nanoscale molecular dynamics with improved reliability and simplicity.

Main Methods:

  • Utilized lanthanide-doped upconversion nanoparticles (UCNPs) with tunable red-to-green (R/G) emission ratios.
  • Investigated the mechanism of ratiometric behavior governed by Ytterbium (Yb3+) excitation density and Erbium (Er3+) ion energy levels.
  • Established a quantitative relationship between UCNP structure and R/G emission ratio for predictable spectral output.

Main Results:

  • Achieved over 10-fold tunability in the R/G emission ratio of UCNPs.
  • Demonstrated simultaneous five-color single-particle imaging with a misidentification rate below 5%.
  • Successfully visualized receptor-mediated endocytosis in live cells using the developed strategy.

Conclusions:

  • Upconversion luminescence-based R/G ratio discrimination offers a robust method for long-term, multicolor SPT.
  • This strategy provides a simple, reliable tool for probing complex biological processes at the nanoscale.
  • The developed UCNP-based imaging overcomes limitations of photobleaching and spectral overlap in multicolor tracking.