Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy

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Title: Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy
Author(s): French, PMW
Görlitz, F
Kelly, D
Warren, S
Alibhai, D
West, L
Kumar, S
Alexandrov, Y
Munro, I
McGinty, J
Talbot, C
Serwa, R
Thinon, E
Da Paola, V
Murray, EJ
Stuhmeier, F
Neil, M
Tate, E
Dunsby, C
Item Type: Journal Article
Abstract: 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 here the 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.
Publication Date: 18-Jan-2017
Date of Acceptance: 13-Sep-2016
URI: http://hdl.handle.net/10044/1/40884
ISSN: 1940-087X
Publisher: Journal of Visualized Experiments (JoVE)
Journal / Book Title: Jove-Journal of Visualized Experiments
Copyright Statement: This paper is embargoed until publication.
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Wellcome Trust
Engineering & Physical Science Research Council (E
Biotechnology and Biological Sciences Research Council (BBSRC)
Funder's Grant Number: BB/H00713X/1
295931/Z/11/Z
EP/K503733/1
BB/M006786/1
Publication Status: Accepted
Embargo Date: publication subject to indefinite embargo
Appears in Collections:Physics
Chemistry
Biological and Biophysical Chemistry
Photonics
Faculty of Natural Sciences



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