期刊
JOURNAL OF THE ROYAL SOCIETY INTERFACE
卷 6, 期 -, 页码 S93-S105出版社
ROYAL SOC
DOI: 10.1098/rsif.2008.0451.focus
关键词
fluorescence lifetime; time-correlated single-photon counting; time-domain fluorescence lifetime imaging microscopy; Forster resonance energy transfer; global fitting
资金
- Cancer Research UK [C133/A/1812]
- UK Research Councils Basic Technology Programme [GR/R87901/01]
- EPSRC [EP/C546113/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/C546113/1] Funding Source: researchfish
Forster 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.
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