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Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (...

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Detalles Bibliográficos
Autores principales: Hum, Julia M., Siegel, Amanda P., Pavalko, Fredrick M., Day, Richard N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Diversity Preservation International (MDPI) 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509586/
https://www.ncbi.nlm.nih.gov/pubmed/23203070
http://dx.doi.org/10.3390/ijms131114385
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author Hum, Julia M.
Siegel, Amanda P.
Pavalko, Fredrick M.
Day, Richard N.
author_facet Hum, Julia M.
Siegel, Amanda P.
Pavalko, Fredrick M.
Day, Richard N.
author_sort Hum, Julia M.
collection PubMed
description Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.
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spelling pubmed-35095862013-01-09 Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy Hum, Julia M. Siegel, Amanda P. Pavalko, Fredrick M. Day, Richard N. Int J Mol Sci Review Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail. Molecular Diversity Preservation International (MDPI) 2012-11-07 /pmc/articles/PMC3509586/ /pubmed/23203070 http://dx.doi.org/10.3390/ijms131114385 Text en © 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0).
spellingShingle Review
Hum, Julia M.
Siegel, Amanda P.
Pavalko, Fredrick M.
Day, Richard N.
Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title_full Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title_fullStr Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title_full_unstemmed Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title_short Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
title_sort monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509586/
https://www.ncbi.nlm.nih.gov/pubmed/23203070
http://dx.doi.org/10.3390/ijms131114385
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