Assessing FRET using spectral techniques.
Assessing FRET using spectral techniques.
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DOI：
10.1002/cyto.a.22340
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发表时间：
2013-10
期刊：
影响因子：
3.7
通讯作者：
Rich, Thomas C.
中科院分区：
文献类型：
--
作者：
Leavesley, Silas J.;Britain, Andrea L.;Cichon, Lauren K.;Nikolaev, Viacheslav O.;Rich, Thomas C.
Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein–protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP–Epac–YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions.
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DOI：
10.1074/jbc.m110.203604
发表时间：
2011-04-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Blackman BE;Horner K;Heidmann J;Wang D;Richter W;Rich TC;Conti M
通讯作者：
Conti M
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
影响因子：
4
作者：
Padilla-Parra, Sergi;Tramier, Marc
通讯作者：
Tramier, Marc
影响因子：
3.8
作者：
Fereidouni, Farzad;Bader, Arjen N.;Gerritsen, Hans C.
通讯作者：
Gerritsen, Hans C.
影响因子：
3.4
作者：
Gordon, GW;Berry, G;Herman, B
通讯作者：
Herman, B