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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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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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Tagging and Fusion Proteins01:24

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Related Experiment Video

Updated: Jul 17, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag.

Soohyen Jang1,2, Kaarjel K Narayanasamy1,3, Johanna V Rahm1

  • 1Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.

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Summary

This study introduces a faster single-molecule localization microscopy technique using a neural network and a novel protein label. This method overcomes limitations of long acquisition times and photobleaching for live-cell imaging.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Single-molecule localization microscopy (SMLM) offers high spatial resolution but suffers from long acquisition times and photobleaching.
  • These limitations hinder the study of dynamic cellular processes in live cells due to low throughput and signal degradation.

Purpose of the Study:

  • To develop an SMLM approach that significantly reduces acquisition time and overcomes photobleaching.
  • To enable high-temporal-resolution live-cell imaging of cellular dynamics.

Main Methods:

  • Integration of the DeepSTORM neural network to predict super-resolution images from high-density data, accelerating image acquisition.
  • Utilizing HaloTag7 protein labeling with exchangeable ligands (xHTLs) for continuous fluorescence signal and photobleaching resistance.
  • Combining neural network prediction with a direct protein label for enhanced live-cell imaging.

Main Results:

  • Achieved up to a ~25-fold reduction in acquisition time compared to conventional SMLM.
  • Demonstrated significantly improved temporal resolution for live-cell imaging.
  • Successfully captured endoplasmic reticulum dynamics over extended periods without signal loss.

Conclusions:

  • The combined approach of DeepSTORM and HaloTag7/xHTLs effectively addresses major SMLM challenges.
  • This method significantly enhances throughput and temporal resolution for live-cell super-resolution microscopy.
  • Enables long-term observation of dynamic cellular events with high fidelity.