[22] Fluorescence lifetime-resolved imaging: Measuring lifetimes in an image

Clegg, Robert M.; Holub, Oliver; Gohlke, Christopher

doi:10.1016/s0076-6879(03)60126-6

Cited by 92 publications

(63 citation statements)

References 67 publications

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“…FRET is a nonradiative energy transfer that can occur when a donor and a compatible acceptor fluorophore are located at a distance lower than 10 nm from each other (15). FRET can be detected via tdFLIM and used to monitor protein-protein interactions in living cells (11). Indeed, if the interacting proteins are conjugated to suitable donor and acceptor fluorophores in such a way that the fluorescence emission spectrum of the donor overlaps the absorption spectrum of the acceptor and their dipoles align, FRET occurs and reduces the lifetime of the donor fluorescence (11).…”

Section: Ebna-1 and Ebp2 Partly Colocalize In Living Cells Throughoutmentioning

confidence: 99%

“…FRET can be detected via tdFLIM and used to monitor protein-protein interactions in living cells (11). Indeed, if the interacting proteins are conjugated to suitable donor and acceptor fluorophores in such a way that the fluorescence emission spectrum of the donor overlaps the absorption spectrum of the acceptor and their dipoles align, FRET occurs and reduces the lifetime of the donor fluorescence (11). In the present case, a tdFLIM setup was used to measure FRET with a spatial accuracy of a few hundred nanometers, which is therefore well adapted for studies at a subcellular scale.…”

Section: Ebna-1 and Ebp2 Partly Colocalize In Living Cells Throughoutmentioning

confidence: 99%

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Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

Jourdan

Jobart-Malfait

Reis

et al. 2012

J Virol

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e Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast doublehybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.

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Section: Ebna-1 and Ebp2 Partly Colocalize In Living Cells Throughoutmentioning

confidence: 99%

Section: Ebna-1 and Ebp2 Partly Colocalize In Living Cells Throughoutmentioning

confidence: 99%

Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

Jourdan

Jobart-Malfait

Reis

et al. 2012

J Virol

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“…In contrast to the sensitized emission approach, FLIM is less dependent on the expression level of the donoracceptor pair because the lifetime is independent of the number of fluorescing molecules. Changes in the lifetime can be solely attributed to changes in the environment of the donor fluorophore, such as proximity to the acceptor, ion concentrations, and temperature (35). Cells were fixed after 20 h and imaged by FLIM.…”

Section: Construction Of a Fret-based Reporter Of Mark Activity In Cementioning

confidence: 99%

Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

Timm

Kries

et al. 2011

Journal of Biological Chemistry

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Background: Deregulation of the protein kinase MARK has been linked to Alzheimer disease. Results: Mark-specific inhibitors and a biosensor are identified. Conclusion:The inhibitors and the biosensor are tools to provide new insights into the role of MARK during polarity establishment and maintenance of neurons. Significance: The inhibitors might possess therapeutic potential by interfering with abnormal Tau phosphorylation in Alzheimer disease.

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“…Fluorescence lifetime imaging (FLIM), which spatially maps the donor lifetime [8], can overcome the problems of intensity-based methods because the lifetime is independent of the number of fluorescing molecules, so changes in lifetime reflect changes in the environment of the probe (e.g. proximity of an acceptor chromophore, ion concentration or temperature).…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Millington

Grindlay

Altenbach

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT High-Precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion proteinMichael Millington, [a, b] G. Joan Grindlay, [c] Kirsten Altenbach, [a, d] Robert K. Neely, [a, b] Walter Kolch, [ c, e] Mojca Bencina , [ f ] Nick D. Read, [a, d] Anita C. Jones, [a, b] David T. F.Dryden, [a, b]

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[22] Fluorescence lifetime-resolved imaging: Measuring lifetimes in an image

Cited by 92 publications

References 67 publications

Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

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