Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis
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...
| Príomhchruthaitheoirí: | , , , , , , |
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| Formáid: | Journal article |
| Teanga: | English |
| Foilsithe / Cruthaithe: |
2009
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| _version_ | 1826263766112665600 |
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| author | Barber, P Ameer-Beg, S Gilbey, J Carlin, L Keppler, M Ng, T Vojnovic, B |
| author_facet | Barber, P Ameer-Beg, S Gilbey, J Carlin, L Keppler, M Ng, T Vojnovic, B |
| author_sort | Barber, P |
| collection | OXFORD |
| description | 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. © 2008 The Royal Society. |
| first_indexed | 2024-03-06T19:57:03Z |
| format | Journal article |
| id | oxford-uuid:25f92c85-4d22-47c8-90b7-d41c3f86f8d5 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T19:57:03Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | oxford-uuid:25f92c85-4d22-47c8-90b7-d41c3f86f8d52022-03-26T11:58:26ZMultiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysisJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:25f92c85-4d22-47c8-90b7-d41c3f86f8d5EnglishSymplectic Elements at Oxford2009Barber, PAmeer-Beg, SGilbey, JCarlin, LKeppler, MNg, TVojnovic, BFörster 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. © 2008 The Royal Society. |
| spellingShingle | Barber, P Ameer-Beg, S Gilbey, J Carlin, L Keppler, M Ng, T Vojnovic, B Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title | Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title_full | Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title_fullStr | Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title_full_unstemmed | Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title_short | Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis |
| title_sort | multiphoton time domain fluorescence lifetime imaging microscopy practical application to protein protein interactions using global analysis |
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