Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis

Barber, P.R., Ameer-Beg, S.M., Gilbey, J., Carlin, L.M. , Keppler, M., Ng, T.C. and Vojnovic, B. (2009) Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis. Journal of the Royal Society: Interface, 6(Suppl1), S93-S105. (doi:10.1098/rsif.2008.0451.focus) (PMCID:PMC2706460)

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Abstract

Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein–protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg–Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.

Item Type:Articles
Additional Information:We acknowledge the support of Cancer Research UK under programme grant C133/A/1812 and of the UK Research Councils Basic Technology Programme under grant GR/R87901/01.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Carlin, Dr Leo
Authors: Barber, P.R., Ameer-Beg, S.M., Gilbey, J., Carlin, L.M., Keppler, M., Ng, T.C., and Vojnovic, B.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Journal of the Royal Society: Interface
Publisher:Royal Society
ISSN:1742-5689
ISSN (Online):1742-5662
Published Online:09 December 2008

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