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Related Concept Videos

Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
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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.
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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Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime

Tobias Grothe1,2, Peter J Walla3,4

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Fluorescence Cross-Correlation Spectroscopy (FCCS) provides a single-molecule view for studying biochemical reactions and molecular dynamics. This guide details setting up an FCCS tool for biological research, enhancing its application in biophysics.

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Building a spectroscopeFluorescence cross-correlation spectroscopyFluorescence lifetime analysisTwo-photon excitation

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Area of Science:

  • Biophysics
  • Molecular Biochemistry
  • Physical Chemistry

Background:

  • Fluorescence Cross-Correlation Spectroscopy (FCCS) is a powerful technique bridging bulk and single-molecule analysis.
  • Its applications span chemical kinetics, protein interactions, conformational changes, and intracellular transport.
  • FCCS is adept at characterizing complex biological structures like lipid vesicles and protein complexes.

Purpose of the Study:

  • To provide a detailed, step-by-step guide for setting up an FCCS instrument.
  • To illustrate the versatility of FCCS, particularly when combined with fluorescence lifetime analysis.
  • To enable researchers to apply FCCS for solving diverse biological problems.

Main Methods:

  • Utilizing a two-photon excitation source for ease of use and maintenance.
  • Integrating fluorescence lifetime analysis with FCCS.
  • Detailed procedural instructions for instrument setup.

Main Results:

  • Demonstrates the capability of FCCS to monitor subtle environmental changes (pH, salt concentration) using appropriate fluorophores.
  • Highlights FCCS's utility in membrane docking and fusion assays.
  • Provides a practical framework for implementing FCCS in biological research.

Conclusions:

  • FCCS is a versatile and accessible tool for advanced biophysical studies.
  • The described setup facilitates the investigation of dynamic biological processes at the molecular level.
  • Combining FCCS with fluorescence lifetime analysis expands its problem-solving capacity in biochemistry.