BibTex format
@article{French:2017:10.3791/55119,
author = {French, PMW and Görlitz, F and Kelly, D and Warren, S and Alibhai, D and West, L and Kumar, S and Alexandrov, Y and Munro, I and McGinty, J and Talbot, C and Serwa, R and Thinon, E and Da, Paola V and Murray, EJ and Stuhmeier, F and Neil, M and Tate, E and Dunsby, C},
doi = {10.3791/55119},
journal = {Jove-Journal of Visualized Experiments},
title = {Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy},
url = {http://dx.doi.org/10.3791/55119},
volume = {119},
year = {2017}
}
RIS format (EndNote, RefMan)
TY - JOUR
AB - We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
AU - French,PMW
AU - Görlitz,F
AU - Kelly,D
AU - Warren,S
AU - Alibhai,D
AU - West,L
AU - Kumar,S
AU - Alexandrov,Y
AU - Munro,I
AU - McGinty,J
AU - Talbot,C
AU - Serwa,R
AU - Thinon,E
AU - Da,Paola V
AU - Murray,EJ
AU - Stuhmeier,F
AU - Neil,M
AU - Tate,E
AU - Dunsby,C
DO - 10.3791/55119
PY - 2017///
SN - 1940-087X
TI - Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy
T2 - Jove-Journal of Visualized Experiments
UR - http://dx.doi.org/10.3791/55119
UR - http://hdl.handle.net/10044/1/40884
VL - 119
ER -