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Protein Dynamics in Living Cells01:19

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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Related Experiment Video

Updated: Jul 12, 2026

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
15:10

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Published on: October 9, 2014

Imaging diffusion in living cells using time-correlated single-photon counting.

Christian M Roth1, Pia I Heinlein, Mike Heilemann

  • 1Physikalisch-Chemisches Insitut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany.

Analytical Chemistry
|September 7, 2007
PubMed
Summary

Diffusion imaging microscopy offers a new way to map protein movement in living cells. This technique provides spatially resolved diffusion times, overcoming limitations of current methods.

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Last Updated: Jul 12, 2026

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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

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Area of Science:

  • Cell biology
  • Biophysics
  • Microscopy

Background:

  • Current methods like fluorescence correlation spectroscopy (FCS) lack spatial resolution for diffusion mapping.
  • Monitoring protein diffusion is crucial for understanding cellular processes.

Purpose of the Study:

  • Introduce diffusion imaging microscopy (DIM) for spatially resolved diffusion time measurements.
  • Overcome limitations of existing techniques in mapping cellular diffusion.

Main Methods:

  • Combine scanning confocal microscopy, time-correlated single-photon counting, and FCS.
  • Record time-resolved photon streams per pixel with extended dwell times.
  • Utilize software correlation of photons within pixels to determine diffusion times.

Main Results:

  • Successfully measured diffusion times in solutions of varying viscosity.
  • Demonstrated applicability to living cells, generating a diffusion map of a mouse fibroblast.
  • Collected fluorescence intensity and lifetime data for photon sorting and autofluorescence discrimination.

Conclusions:

  • Diffusion imaging microscopy enables spatially resolved measurement of protein diffusion times.
  • This novel method provides valuable insights into cellular dynamics and protein transport.
  • DIM offers a significant advancement for studying molecular diffusion in complex biological systems.