Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane type 1 matrix metalloproteinase in live cells

John Paul Eichorst; He Huang; Robert M. Clegg; Yingxiao Wang

doi:10.1007/s10895-011-0871-x

Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane type 1 matrix metalloproteinase in live cells

John Paul Eichorst, He Huang, Robert M. Clegg, Yingxiao Wang

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with fullfield frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.

Original language	English (US)
Pages (from-to)	1763-1777
Number of pages	15
Journal	Journal of Fluorescence
Volume	21
Issue number	4
DOIs	https://doi.org/10.1007/s10895-011-0871-x
State	Published - Jul 2011
Externally published	Yes

Bibliographical note

Funding Information:
Acknowledgements This work is supported in part by grants from NIH HL098472, CA139272, NS063405, NSF CBET0846429, CMMI0800870, the Wallace H. Coulter Foundation and Beckman Laser Institute, Inc. (Y.W.). We thank ISS for the use of the cuvette-based lifetime equipment.

Keywords

Biosensors
Fluorescence Lifetime Imaging Microscopy-FLIM
Fluorescent proteins
Frequency-domain
Förster resonance energy transfer-FRET
Membrane proteinase
Phase differential enhancement
Phase suppression

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Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane type 1 matrix metalloproteinase in live cells. / Eichorst, John Paul; Huang, He; Clegg, Robert M. et al.
In: Journal of Fluorescence, Vol. 21, No. 4, 07.2011, p. 1763-1777.

Research output: Contribution to journal › Article › peer-review

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abstract = "Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with fullfield frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.",

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Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane type 1 matrix metalloproteinase in live cells

Abstract

Bibliographical note

Keywords

UN SDGs

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