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Protein Dynamics in Living Cells01:19

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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.
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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Related Experiment Video

Updated: Apr 27, 2026

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

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Imaging fluorescence-correlation spectroscopy for measuring fast surface diffusion at liquid/solid interfaces.

Justin T Cooper1, Joel M Harris

  • 1Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0805, United States.

Analytical Chemistry
|July 1, 2014
PubMed
Summary

This study introduces imaging-FCS, a novel technique combining rapid imaging and fluorescence-correlation spectroscopy to measure fast molecular transport at surfaces. It accurately quantifies surface diffusion and adsorption kinetics, crucial for optimizing analytical methods.

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Area of Science:

  • Surface science
  • Analytical chemistry
  • Spectroscopy

Background:

  • Interfacial molecular transport is key for surface-based analytical methods.
  • Existing techniques like single-molecule imaging and FCS have limitations in measuring fast diffusion and handling surface inhomogeneity.

Purpose of the Study:

  • To develop and validate a new technique, imaging-FCS, for measuring fast interfacial molecular transport.
  • To overcome limitations of existing methods for studying surface diffusion and adsorption kinetics.

Main Methods:

  • Combines rapid imaging with fluorescence-correlation spectroscopy (FCS).
  • Utilizes an electron-multiplied CCD camera with controlled acquisition areas for millisecond time resolution.
  • Autocorrelates intensity fluctuations from sampled regions to determine diffusion rates.

Main Results:

  • Successfully measured surface diffusion rates, interfacial populations, and adsorption-desorption kinetics.
  • Demonstrated millisecond time resolution for interfacial transport.
  • Showcased the ability to electronically control acquisition regions to minimize adsorption site interference.

Conclusions:

  • Imaging-FCS is a powerful tool for studying fast interfacial transport.
  • The technique enhances understanding and optimization of surface-based analytical methods.
  • Provides accurate measurements of diffusion and adsorption kinetics on model chromatographic surfaces.