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...

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

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