A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

Abstract

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-S H74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.FRET requires sufficient overlap of excitation spectrum of the acceptor with the emission spectrum of the donor and also depends on the relative angular orientations of the donor and acceptor dipoles [4].In the case of heteroFRET, the two fluorophores are not identical and emit at different wavelengths [5]. There are a very large number of approaches to analysing spectral ratio-metric FRET data [6,7], the simplest of which is a straight-forward ratio of sensitized acceptor emission to the directly excited emission from the donor fluorophore. This simple calculation of the FRET ratio is useful for real-time monitoring of biosensor readouts in live cells and tissue slices.Over recent years, a number of non-proprietary software packages have been written for ratio-metric FRET analysis, however their use is limited to analysis of previously saved image-stacks [8][9][10][11]. Some proprietary software offers real-time measurements of the ratios between fluorescent intensities in two channels; one example is MetaFluor (Molecular Devices, San Jose, USA) [12]. This provides additional flexibility, allowing experimental procedures to be carried out in response to changes in the FRET ratio. As a low-cost alternative, Julia U. Sprenger et al. (2012) built a customised epifluorescence FRET imaging system and developed a non-proprietary ImageJ macro, which displays real-time ratio-metric data obtained from a Micro-Manager controlled time-lapse acquis...

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“…Time-lapse imaging was executed with a Hamamatsu ORCA-Flash4.0 camera (Hamamatsu Photonics UK Ltd, Welwyn Garden City, United Kingdom), with a frame every 6 seconds. The image acquisition and analysis by quantifying the relative ratio of YFP to CFP image intensity in the regions of interest (ROI) was performed using a custom-made plug-in to Icy freeware (http://icy.bioimageanalysis.org/) using the Micro-Manager1.4 (http://micro-manager.org) software package [26].…”

Special Issue on “New Advances in Cyclic AMP Signalling”—An Editorial Overview

“…Thus, for proper physiological function, it is vital that cyclic AMP signalling occurs in nanodomains. A major advance in this Special Edition is the report of a new software tool, MultiFRET, which, together with a mechanical microscope stage, allows the assessment of multiple cells in the same experiment [ 68 ]. This should allow researchers to determine FRET from multiple cells within one specimen and, therefore, very important for producing data from samples where there is a large heterogeneity within the cell population, such as primary cells.…”

“…This should allow researchers to determine FRET from multiple cells within one specimen and, therefore, very important for producing data from samples where there is a large heterogeneity within the cell population, such as primary cells. Therefore, the researchers examined the FRET responses using multiple probe constructs (pmEpac-1, Epac1-PLN, epac1-cyclic AMPs, AKAP79-CUTie, Epac-SH74) in multiple cell types (adult and neonatal mouse cardiomyocytes and hiPSC cells) [ 68 ]. It is envisaged that this approach may be scaled up for high throughput screening in order to identify drugs that target individual nanodomains in a cell population.…”

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“…However, due to the rapid upgrades of microscopy equipment in the market, most existing μManager plugins lack development speedy enough to provide sufficient compatibility with the latest equipment. For example, a recently developed μManager plugin, MultiFRET, can achieve automatic acquisition and analysis of fluorescent images but relies on μManager-supported instruments [ 13 ]. Due to the same restriction, another newly developed plugin, Easier Micro-Manager User (EMU), can offer only limited functions to control μManager-supported optical hardware, e.g., modulation of laser and filter wheels, and acquisition processes, e.g., 2D time series or 3D z-stack imaging [ 14 ].…”