期刊
JOURNAL OF MICROSCOPY
卷 237, 期 1, 页码 51-62出版社
WILEY
DOI: 10.1111/j.1365-2818.2009.03301.x
关键词
Fluorescence anisotropy; FRET; high-content screening; microscopy; time lapse
类别
资金
- UK EPSRC [12 (EP/C546105/1)]
- Wellcome Trust
- Dimbleby Cancer Care Award
- Cancer Research UK [C133/A/1812]
- EPSRC [EP/C546113/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/C546105/1, EP/C546113/1] Funding Source: researchfish
P>We present recent data on dynamic imaging of Rac1 activity in live T-cells. Forster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein-protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Forster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.
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