Measurement of Molecular Mobility with Fluorescence Correlation Spectroscopy

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Abstract

Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to subfemtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments - including the nucleus, the cytoplasm, and the membrane - can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment. Curr. Protoc. Cytom. 50:2.15.1-2.15.19.

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Fluorescence Spectrometry
Fluorescence
Spectroscopy
Molecular interactions
Photochemical reactions
Spectrum Analysis
Fluorescence spectroscopy
Cytoplasm
Color
Autocorrelation
Observation
Labeling
Ligands
Membranes
Antibodies
Association reactions
Molecules
Proteins

Keywords

  • Cellular FCS measurements
  • Co-diffusion
  • Diffusion
  • FCCS
  • FCS
  • Fluorescence correlation spectroscopy
  • Fluorescence cross-correlation spectroscopy
  • Molecular mobility

ASJC Scopus subject areas

  • Medical Laboratory Technology
  • Biochemistry
  • Histology

Cite this

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title = "Measurement of Molecular Mobility with Fluorescence Correlation Spectroscopy",
abstract = "Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to subfemtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments - including the nucleus, the cytoplasm, and the membrane - can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment. Curr. Protoc. Cytom. 50:2.15.1-2.15.19.",
keywords = "Cellular FCS measurements, Co-diffusion, Diffusion, FCCS, FCS, Fluorescence correlation spectroscopy, Fluorescence cross-correlation spectroscopy, Molecular mobility",
author = "G. V{\'a}mosi and S. Damjanovich and J. Sz{\"o}llősi and G. Vereb",
year = "2009",
doi = "10.1002/0471142956.cy0215s50",
language = "English",
journal = "Current Protocols in Cytometry",
issn = "1934-9297",
publisher = "John Wiley and Sons Inc.",
number = "SUPPL.50",

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T1 - Measurement of Molecular Mobility with Fluorescence Correlation Spectroscopy

AU - Vámosi, G.

AU - Damjanovich, S.

AU - Szöllősi, J.

AU - Vereb, G.

PY - 2009

Y1 - 2009

N2 - Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to subfemtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments - including the nucleus, the cytoplasm, and the membrane - can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment. Curr. Protoc. Cytom. 50:2.15.1-2.15.19.

AB - Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to subfemtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments - including the nucleus, the cytoplasm, and the membrane - can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment. Curr. Protoc. Cytom. 50:2.15.1-2.15.19.

KW - Cellular FCS measurements

KW - Co-diffusion

KW - Diffusion

KW - FCCS

KW - FCS

KW - Fluorescence correlation spectroscopy

KW - Fluorescence cross-correlation spectroscopy

KW - Molecular mobility

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