Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

Mark A A Neil,Yuriy Alexandrov,Clifford Talbot,Paul M W French,James Mcginty,Emmanuelle Thinon,Ian Munro,Lucien West,Dominic Alibhai,Christopher Dunsby,Frederik Görlitz,Remigiusz A Serwa,Vincenzo Da Paola,Sunil Kumar,Edward W Tate,Frank Stühmeier ,Edwin García ,Sean Warren ,Edward J Murray ,Douglas J Kelly

doi:10.3791/55119

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 µManager. 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.

Highlights

The increasing use of automated high content analysis (HCA) in drug discovery[1] to assay compound libraries is complemented by the trend in life science basic research towards automated imaging experiments for systematic studies, e.g., for phenotypic screens of siRNA libraries
To illustrate the capabilities of the openFLIM-HCA instrumentation, we present three exemplar Förster resonance energy transfer (FRET) applications
To provide a negative control, the Venus in the short 5-amino acid linker construct is replaced by Amber — a non-fluorescent mutation of YFP that cannot act as a FRET acceptor[38]

Summary

Introduction

The increasing use of automated high content analysis (HCA) in drug discovery[1] to assay compound libraries is complemented by the trend in life science basic research towards automated imaging experiments for systematic studies, e.g., for phenotypic screens of siRNA libraries. Our implementation of FLIM HCA comprises six key components: (i) a fluorescence microscope frame with optical autofocus; (ii) a motorized (x-y-z) microscope stage; (iii) an excitation laser source; (iv) a wide-field time-gated FLIM system with gated optical intensifier (GOI), delay generator and camera; (v) a computer for data acquisition and analysis and (vi) a Nipkow disc scanner unit for optically sectioned imaging to suppress out of focus light This can be important when imaging weak signals, e.g., from FRET biosensors at the cell plasma membrane, that could be swamped by contributions from cytoplasmic fluorescence or background from coverslips or culture medium.

Preparation of Multiwell Plate Array

11. Acquire IRF image data

12. Acquire reference dye data

Representative Results

Discussion