Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 14385-14400 |
| Number of pages | 16 |
| Journal | International Journal of Molecular Sciences |
| Volume | 13 |
| Issue number | 11 |
| DOIs | |
| State | Published - 2012 |
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Keywords
- Biosensor probe
- Cell signaling
- Fluorescence lifetime imaging microscopy (FLIM)
- Fluorescence resonance energy transfer (FRET)
- Fluorescent protein
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Organic Chemistry
- Spectroscopy
- Inorganic Chemistry
- Catalysis
- Molecular Biology
- Computer Science Applications
- Medicine(all)
Cite this
Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy. / Hum, Julia M.; Siegel, Amanda P.; Pavalko, Fredrick; Day, Richard.
In: International Journal of Molecular Sciences, Vol. 13, No. 11, 2012, p. 14385-14400.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy
AU - Hum, Julia M.
AU - Siegel, Amanda P.
AU - Pavalko, Fredrick
AU - Day, Richard
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - Biosensor probe
KW - Cell signaling
KW - Fluorescence lifetime imaging microscopy (FLIM)
KW - Fluorescence resonance energy transfer (FRET)
KW - Fluorescent protein
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UR - http://www.scopus.com/inward/citedby.url?scp=84870652819&partnerID=8YFLogxK
U2 - 10.3390/ijms131114385
DO - 10.3390/ijms131114385
M3 - Article
C2 - 23203070
AN - SCOPUS:84870652819
VL - 13
SP - 14385
EP - 14400
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1661-6596
IS - 11
ER -