Time-lapse FRET microscopy using fluorescence anisotropy.
We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster 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 Förster 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.
Matthews
D R
DR
Richard Dimbleby Department of Cancer Research, New Hunts House, Kings College London, Guy's Medical School Campus, SE11UL, UK. daniel.matthews@kcl.ac.uk
Carlin
L M
LM
Ofo
E
E
Barber
P R
PR
Vojnovic
B
B
Irving
M
M
Ng
T
T
Ameer-Beg
S M
SM
D015374:Biosensing Techniques; D002460:Cell Line; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000737:chemistry; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000737:chemistry; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D000097573:Red Fluorescent Protein; C107953:RAC1 protein, human