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Related Concept Videos

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...
Protein Diffusion in the Membrane01:24

Protein Diffusion in the Membrane

Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...

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Related Experiment Video

Updated: May 19, 2026

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer
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Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise.

Fabian Heinemann1, Viktoria Betaneli, Franziska A Thomas

  • 1Biophysics Institute, Biotec/Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany.

Langmuir : the ACS Journal of Surfaces and Colloids
|August 16, 2012
PubMed
Summary

This study establishes a reliable reference diffusion coefficient for the fluorescent lipid DiD in DOPC membranes using five Fluorescence Correlation Spectroscopy (FCS) variants. The consistent results across methods validate its use for calibrating membrane diffusion measurements.

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Last Updated: May 19, 2026

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Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
10:59

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Published on: April 6, 2012

Area of Science:

  • Membrane biophysics
  • Spectroscopy
  • Physical chemistry

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is crucial for measuring diffusion in biological membranes.
  • Existing FCS methods lack comprehensive comparison and consistent reference values for membrane systems.
  • This hinders accurate determination of lipid and protein diffusion coefficients.

Purpose of the Study:

  • To establish a reliable reference diffusion coefficient for the fluorescent lipid analog DiD in DOPC GUVs.
  • To compare five different FCS variants and their calibration methods.
  • To improve the comparability and accuracy of FCS experiments on biological membranes.

Main Methods:

  • Utilized five distinct Fluorescence Correlation Spectroscopy (FCS) variants.
  • Employed different calibration strategies within the FCS methods.
  • Measured diffusion of the fluorescent lipid DiD in giant unilamellar vesicles (GUVs) composed of DOPC and DOPC/cholesterol.

Main Results:

  • Obtained consistent absolute diffusion coefficients for DiD in DOPC across all five FCS variants.
  • Determined an average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C.
  • Verified method comparability for both fast and slow diffusion scenarios.

Conclusions:

  • The determined DiD diffusion coefficient in DOPC GUVs serves as a valuable reference standard.
  • The study validates the use of this value for calibrating future FCS membrane experiments.
  • Demonstrated the reliability and comparability of multiple FCS variants for membrane diffusion studies.