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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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Updated: Jul 1, 2026

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Published on: February 1, 2017

Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy.

You Korlann¹, Thomas Dertinger, Xavier Michalet

¹Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.

|September 17, 2008

Summary

This summary is machine-generated.

We introduce polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS) for measuring molecular diffusion coefficients. This new method is simpler than existing techniques and accurate even with high laser power.

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Last Updated: Jul 1, 2026

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

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Published on: February 1, 2017

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

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Published on: November 12, 2020

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Published on: December 11, 2021

Area of Science:

Biophysics
Spectroscopy
Physical Chemistry

Background:

Fluorescence Correlation Spectroscopy (FCS) is crucial for studying molecular dynamics.
Conventional FCS struggles with optical saturation, affecting diffusion coefficient accuracy.
Dual-focus FCS (2fFCS) offers improved accuracy but can be complex to implement.

Purpose of the Study:

To develop a simpler and robust method for measuring diffusion coefficients.
To overcome optical saturation limitations in fluorescence spectroscopy.
To present polarization-modulation dual-focus FCS (pmFCS) as an accessible technique.

Main Methods:

Implementation of pmFCS on a conventional confocal microscope.
Utilizing an electro-optical modulator and a differential interference contrast prism.
Measuring diffusion coefficients of fluorescent molecules at low concentrations.

Main Results:

pmFCS provides accurate diffusion coefficients, unaffected by optical saturation.
The technique demonstrated robustness comparable to 2fFCS.
A diffusion coefficient of (4.09 ± 0.07) x 10⁻⁶ cm²/s was measured for Atto655 maleimide in water at 25°C.

Conclusions:

pmFCS is a simpler, more accessible alternative to 2fFCS for diffusion coefficient measurements.
The technique maintains accuracy under conditions where conventional FCS fails.
pmFCS is compatible with standard confocal microscopy setups and continuous-wave excitation.