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Related Concept Videos

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...
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.
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,...
Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

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Total Internal Reflection Fluorescence Microscopy

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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Fluorescence correlation spectroscopy in semiadhesive wall proximity.

Luigi Sanguigno1, Ilaria De Santo, Filippo Causa

  • 1Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Naples, Italy. luigi.sanguigno@iit.it

Analytical Chemistry
|September 22, 2011
PubMed
Summary

Anomalous diffusion in heterogeneous media is often poorly understood. This study introduces a new model for fluorescence correlation spectroscopy that accounts for obstacles, improving the analysis of diffusion in complex biological systems.

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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)

Published on: April 6, 2012

Area of Science:

  • Physical Chemistry
  • Biophysics
  • Chemical Physics

Background:

  • Fluorescence correlation spectroscopy (FCS) in heterogeneous media often exhibits anomalous diffusion, characterized by long correlation tails.
  • Standard diffusion models struggle to accurately determine diffusion times due to excluded volume and sticking effects.
  • Anomalous diffusion coefficients provide general explanations but lack specificity for underlying mechanisms.

Purpose of the Study:

  • To investigate if inadequate models for heterogeneous systems contribute to observed diffusion anomalies in FCS.
  • To explore the impact of obstacles within the detection volume on FCS experiments.
  • To develop a refined model for analyzing diffusion in complex, confined environments.

Main Methods:

  • Developed an original autocorrelation function model for FCS.
  • Incorporated fluorophore reflection and adsorption at a wall within the detection volume.
  • Analyzed long-time correlations to differentiate steric and adhesion effects.

Main Results:

  • Successfully discriminated between steric hindrance and adhesion effects.
  • Quantified adhesion strength by evaluating adsorption probability and persistence time.
  • Demonstrated the model's ability to interpret complex diffusion behaviors.

Conclusions:

  • The proposed model offers a more precise interpretation of diffusion anomalies in heterogeneous systems.
  • This approach enhances the understanding of intracellular and nanoconfined diffusion.
  • Facilitates more rational analysis of diffusion mechanisms for biological and biomedical applications.