scholarly journals Quantitative imaging of protein–protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera

2003 ◽  
Vol 8 (3) ◽  
pp. 362 ◽  
Author(s):  
R. V. Krishnan ◽  
A. Masuda ◽  
V. E. Centonze ◽  
B. Herman
Keyword(s):  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Streak Camera ◽  
Quantitative Imaging ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging

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.

scholarly journals Photoswitching FRET to monitor protein–protein interactions

2018 ◽  
Vol 116 (3) ◽  
pp. 864-873 ◽  
Author(s):  
Kristin H. Rainey ◽  
George H. Patterson
Keyword(s):  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging ◽  
Powerful Approach ◽  
Fluorescent Molecules ◽  
In Cells

FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule’s photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched “off” rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched “on” again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein–protein interactions and fluorescent protein-based biosensors.

Microarray analysis of protein–protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime imaging

2008 ◽  
Vol 24 (3) ◽  
pp. 397-402 ◽  
Author(s):  
Stefan Nagl ◽  
Reinhard Bauer ◽  
Ursula Sauer ◽  
Claudia Preininger ◽  
Udo Bogner ◽  
...  
Keyword(s):  
Microarray Analysis ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging
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