An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions

2011 ◽  
Author(s):  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Douglas Kelly ◽  
Sean Warren ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging ◽  
Plate Reader ◽  
High Content Analysis

scholarly journals High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis

2008 ◽  
Vol 1 (6) ◽  
pp. 514-521 ◽  
Author(s):  
Clifford B. Talbot ◽  
James McGinty ◽  
David M. Grant ◽  
Ewan J. McGhee ◽  
Dylan M. Owen ◽  
...  
Keyword(s):  
Content Analysis ◽  
Fluorescence Lifetime ◽  
High Speed ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Plate Reader ◽  
High Content Analysis

Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

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

2008 ◽  
Vol 6 (suppl_1) ◽  
Author(s):  
P.R Barber ◽  
S.M Ameer-Beg ◽  
J Gilbey ◽  
L.M Carlin ◽  
M Keppler ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Time Domain ◽  
Single Photon ◽  
Global Analysis ◽  
Exponential Fitting ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging

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.

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