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

Masoud Ramuz,Sian Harding,Julia Gorelik,Alveera Hasan,Lena Gruscheski,Chris Dunsby,Ivan Diakonov,Viacheslav O Nikolaev,Nikoleta Pavlaki

doi:10.3390/cells8121541

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-SH74 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.

Highlights

One of the major experimental methods in cellular biology for investigating the interactions, dynamics and localisation of specific molecules is through the measurement of luminescent or fluorescent probes [1]
As a routine FörsterResonance Energy Transfer (FRET) experiment requires time-lapse acquisition of one frame every few seconds, we wished to increase the throughput by rapidly cycling between several preselected cells in the same dish
We have developed a new open-source Java plugin that increases the speed and ease of real-time FRET data acquisition in live cells

Summary

Introduction

One of the major experimental methods in cellular biology for investigating the interactions, dynamics and localisation of specific molecules is through the measurement of luminescent or fluorescent probes [1]. The luminescent/fluorescent molecule either is attached to a molecule of interest, allowing characterisation of its location and function, or can be used in a sensor molecule that reports a specific local environmental parameter. An example of the latter technique is intermolecular Förster. The donor may transfer its energy non-radiatively to a second acceptor fluorophore if they are separated by less than ~10 nm. The efficiency of energy transfer is inversely proportional to the sixth power of the distance between the two fluorophores [2,3].

Methods

Results

Conclusion