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

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.

[1]  Jun Chu,et al.  A Guide to Fluorescent Protein FRET Pairs , 2016, Sensors.

[2]  B. Corry,et al.  ExiFRET: flexible tool for understanding FRET in complex geometries. , 2012, Journal of biomedical optics.

[3]  Julia Sprenger,et al.  Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET , 2016, PloS one.

[4]  J. Szöllősi,et al.  Understanding FRET as a Research Tool for Cellular Studies , 2015, International journal of molecular sciences.

[5]  Martin J. Lohse,et al.  Novel Single Chain cAMP Sensors for Receptor-induced Signal Propagation*♦ , 2004, Journal of Biological Chemistry.

[6]  S. Lehnart,et al.  Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of β-adrenoceptor-stimulated contractility in early cardiac hypertrophy. , 2015, Circulation research.

[7]  S. Lehnart,et al.  In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease , 2015, Nature Communications.

[8]  R. Fischmeister,et al.  Cyclic AMP signaling in cardiac myocytes , 2018 .

[9]  M. Lohse,et al.  FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells , 2011, Nature Protocols.

[10]  S. Padilla-Parra,et al.  FRET microscopy in the living cell: Different approaches, strengths and weaknesses , 2012, BioEssays : news and reviews in molecular, cellular and developmental biology.

[11]  P. Burridge,et al.  Improved Human Embryonic Stem Cell Embryoid Body Homogeneity and Cardiomyocyte Differentiation from a Novel V‐96 Plate Aggregation System Highlights Interline Variability , 2007, Stem cells.

[12]  Konrad R. Götz,et al.  FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors. , 2012, Journal of visualized experiments : JoVE.

[13]  János Roszik,et al.  Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution , 2009, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[14]  T. Főrster,et al.  10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation , 1959 .

[15]  L. Hodgson,et al.  Quantitative ratiometric imaging of FRET-biosensors in living cells. , 2013, Methods in cell biology.

[16]  G. Drummen.  Fluorescent Probes and Fluorescence (Microscopy) Techniques — Illuminating Biological and Biomedical Research , 2012, Molecules.

[17]  M. Zaccolo,et al.  FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility , 2017, Nature Communications.

[18]  E. Rees,et al.  A quantitative protocol for intensity-based live cell FRET imaging. , 2014, Methods in molecular biology.

[19]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[20]  D. Stepensky,et al.  FRETcalc plugin for calculation of FRET in non-continuous intracellular compartments. , 2007, Biochemical and biophysical research communications.

[21]  Daniel Sage,et al.  PixFRET, an ImageJ plug‐in for FRET calculation that can accommodate variations in spectral bleed‐throughs , 2005, Microscopy research and technique.

[22]  Joachim Goedhart,et al.  A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range , 2011, PloS one.