FLIM-FRET analyzer: open source software for automation of lifetime-based FRET analysis
© The Author(s). 2017
Received: 11 January 2017
Accepted: 24 October 2017
Published: 3 November 2017
Despite the broad use of FRET techniques, available methods for analyzing protein-protein interaction are subject to high labor and lack of systematic analysis. We propose an open source software allowing the quantitative analysis of fluorescence lifetime imaging (FLIM) while integrating the steady-state fluorescence intensity information for protein-protein interaction studies.
Our developed open source software is dedicated to fluorescence lifetime imaging microscopy (FLIM) data obtained from Becker & Hickl SPC-830. FLIM-FRET analyzer includes: a user-friendly interface enabling automated intensity-based segmentation into single cells, time-resolved fluorescence data fitting to lifetime value for each segmented objects, batch capability, and data representation with donor lifetime versus acceptor/donor intensity quantification as a measure of protein-protein interactions.
The FLIM-FRET analyzer software is a flexible application for lifetime-based FRET analysis. The application, the C#. NET source code, and detailed documentation are freely available at the following URL: http://FLIM-analyzer.ip-korea.org.
Over the past decades, light microscopy has been customized to facilitate the investigation of protein assembly into macromolecular complexes in living cells. Calculating the efficiency of the Förster/fluorescence resonance energy transfer (FRET) allows characterization of protein-protein interactions or changes in protein conformation, because the efficiency varies as the inverse sixth power of distance between fluorophores, typically reaching 50% at 2–8 nm . Among the few existing approaches toward detecting and quantifying FRET, fluorescent lifetime imaging (FLIM) has a number of advantages. The changes in fluorescence lifetime are independent of fluorophore concentration, simplifying the process of filtering out artifacts introduced by variations in fluorophore concentration and emission intensity across the sample. In contrast to intensity-based FRET measurements, FLIM measurements are relatively robust under conditions where spectral crosstalk is present. As a result, FLIM experiments do not require spectral calibration measurements. Fluorescence lifetime can also be used to distinguish different fluorophores with similar spectral properties  and report variations in the fluorophore’s local environment . Although FLIM is widely used, it is subject to major limitations. A few softwares are available for batching the analysis of FLIM data [4–6], but none provide automated segmentation function for single cell lifetime analysis. Such limitations make FLIM data analysis extremely time intensive, which is a major drawback for protein-protein interaction studies. We therefore have developed a single cell image segmentation software called FLIM-FRET analyzer, which separates objects of interest from the background to delineate whole cells, facilitating image segmentation into single cells followed by donor lifetime and donor/acceptor fluorescence intensity quantification.
Results and discussion
We tested the performance of FLIM-FRET analyzer against that of the standard SPCImage (version 5.4) software, which requires manual segmentation of single cells, using HEK-293 cells expressing fluorescent proteins. Both methods yielded comparable results (Pearson r: 0.9925), suggesting that FLIM-FRET analyzer software is a robust and accurate tool for analyzing protein-protein interactions using fluorescence assays (Additional file 1: Figure S1). Last, we illustrate the application of FLIM-FRET Analyzer upon lifetime quantification of single cell expressing FRET-capable pairs of fluorescent proteins (Additional file 1: Figure S2).
The stand-alone software described here aims to simplify and accelerate the process of analyzing multivariate FLIM data sets for single cell lifetime quantification. The available full C# source code (http://FLIM-analyzer.ip-korea.org.) will allow the user to adapt or extend the currently provided version of the application. A tutorial (Additionnal file 2) can also be downloaded at the same URL.
This work was supported by the National Research foundation of Korea (NRF) grant funded by the Korea government (MSIP)(NRF-2014K1A4A7A01074642; NRF-2017M3A9G6068257), Gyeonggi-do. This work was also funded by the NRF individual scientist support program (NRF-2012R1A1A2004980/ NRF-2015R1D1A1A09057239).
Availability of data and materials
The application, the C#. Net source code, and detailed documentation are freely available at following URL: http://FLIM-analyzer.ip-korea.org
Study conception and design: JK and RG (Leading FLIM-FRET analyzer software development). Acquisition of data: JK (Design the layout of FLIM-FRET analyzer software, supervise the lifetime fitting process, and validate the software), YT (Development and programming) and JP (Develop the software architecture, user interface and workflow). Analysis and interpretation of data: JK, YT and RG. Drafting of manuscript: JK and RG, Critical revision: RG. All authors read and approved the final manuscript.
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