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Gradual labeling with fluorogenic probes: A general method for MINFLUX imaging and tracking

Longfang Yao1,2, Dongjuan Si3, Liwen Chen4

  • 1School of Artificial Intelligence Science and Technology, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China.

Science Advances
|May 21, 2025
PubMed

Insights

We developed gradual labeling with fluorogenic probes for MINFLUX (GLF-MINFLUX) imaging and tracking. This method achieves high-precision live-cell protein tracking and imaging in dense cellular environments.

Area of Science:

  • Biophysics
  • Cell Biology
  • Nanotechnology

Background:

  • Minimal photon fluxes (MINFLUX) nanoscopy is powerful for nanoscale protein studies.
  • Existing MINFLUX methods struggle with simultaneous live-cell imaging and tracking in complex cellular settings.

Purpose of the Study:

  • To develop a universal strategy for simultaneous imaging and live-cell tracking using MINFLUX nanoscopy.
  • To enhance precision, acquisition speed, and target density for nanoscale protein analysis.

Main Methods:

  • Developed gradual labeling with fluorogenic probes for MINFLUX (GLF-MINFLUX).
  • Utilized membrane-permeable, protein-induced 'off/on' switching fluorogenic dyes for gradual labeling, localization, and bleaching.
  • Enabled sequential positioning and tracking of individual proteins.

Main Results:

  • Achieved 2.6-nanometer localization precision for continuous microtubules, outperforming conventional MINFLUX.
  • Demonstrated 1.7-fold improvement in precision, 2.2-fold in acquisition speed, and 3-fold in target density.
  • Enabled 3D localization of mitochondrial proteins and dual-channel imaging of microtubules and microfilaments.
  • Performed live-cell single-protein tracking with 7.8 nm precision at ~200 µs resolution, revealing distinct diffusion behaviors.

Conclusions:

  • GLF-MINFLUX offers a versatile method for nanoscale protein studies by tuning probe concentration.
  • Provides molecular-level insights into protein functions and dynamics in live cells.
  • Significantly advances capabilities for live-cell imaging and tracking in dense cellular environments.

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