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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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

Longfang Yao^1,2, 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

Summary

This summary is machine-generated.

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.

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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.