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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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Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which...
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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
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Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).

Alexander M Mørch1, Falk Schneider2

  • 1Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.

Methods in Molecular Biology (Clifton, N.J.)
|April 27, 2023
PubMed
Summary

Investigating immune cell activation requires understanding fast nanoscale dynamics. Scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) reveal molecular diffusion and interactions during immune synapse formation.

Keywords:
Diffusion dynamicsFFSInteractionsOligomerizationsFCS

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Area of Science:

  • Cell Biology
  • Immunology
  • Biophysics

Background:

  • Immune cell activation and immunological synapse (IS) formation depend on plasma membrane reorganization.
  • These processes involve complex diffusion, oligomerization, and single-molecule interactions.
  • Fast nanoscale dynamics are crucial but often missed by conventional imaging.

Purpose of the Study:

  • To introduce and detail the application of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS).
  • To investigate molecular diffusion dynamics and interactions during IS formation and other biological contexts.
  • To provide practical guidance for implementing sFCS/sFCCS in biological research.

Main Methods:

  • Utilized scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS).
  • Focused on optical setup calibration, alignment, and measurement protocols.
  • Developed data analysis pipelines and strategies for sFCS/sFCCS data.

Main Results:

  • Demonstrated the capability of sFCS/sFCCS to resolve fast nanoscale dynamics in biological systems.
  • Provided a comprehensive protocol for performing and analyzing sFCS/sFCCS measurements.
  • Showcased an application studying Lck diffusion dynamics during T-cell signaling.

Conclusions:

  • sFCS and sFCCS are powerful techniques for studying molecular dynamics at the nanoscale.
  • These methods offer essential insights into the mechanisms of immune cell activation and IS formation.
  • The provided protocol facilitates the adoption of sFCS/sFCCS in diverse biological research areas.