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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...
Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

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.
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
Atomic Fluorescence Spectroscopy01:29

Atomic Fluorescence Spectroscopy

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 are...
Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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.
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Related Experiment Video

Updated: Jul 10, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
14:12

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

Fluorescence correlation spectroscopy in living cells.

Sally A Kim1, Katrin G Heinze, Petra Schwille

  • 1Institute of Biophysics, Dresden University of Technology, Tatzberg 47-51, D-01307 Dresden, Germany.

Nature Methods
|November 1, 2007
PubMed
Summary

Fluorescence correlation spectroscopy (FCS) offers precise molecular analysis in living cells. This guide details FCS methods for accurate cellular studies, minimizing artifacts for reliable molecular dynamics insights.

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

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Last Updated: Jul 10, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

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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:

  • Biophysics
  • Cell Biology
  • Analytical Chemistry

Background:

  • Fluorescence Correlation Spectroscopy (FCS) analyzes fluorescence fluctuations to study molecular dynamics.
  • It is ideal for nanomolar concentrations in living cells, providing insights into diffusion, concentration, and interactions.
  • FCS offers single-molecule sensitivity and high temporal resolution for real-time cellular analysis.

Purpose of the Study:

  • To provide a comprehensive guide for applying FCS to cellular systems.
  • To detail methods for minimizing artifacts and optimizing measurement conditions.
  • To enable accurate parameter extraction in complex intracellular environments.

Main Methods:

  • FCS utilizes autocorrelation analysis of fluorescence intensity fluctuations over time.
  • An observation volume is created using a focused laser beam.
  • Data fitting to physical models yields parameters like diffusion coefficients and concentrations.

Main Results:

  • FCS provides precise information on molecular diffusion, concentration, aggregation, and interactions.
  • The technique achieves diffraction-limited spatial and sub-microsecond temporal resolution.
  • Successful application requires careful optimization to avoid artifacts in cellular measurements.

Conclusions:

  • FCS is a powerful tool for studying molecular dynamics in living cells.
  • This guide addresses challenges and offers solutions for reliable intracellular FCS measurements.
  • Optimized FCS protocols enhance the accuracy of molecular parameter determination within cells.