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