FlickerPrint: An Analysis Package for Measuring Interfacial Tension and Bending Rigidity of Biomolecular Condensates and Vesicles at Scale

1 In BriefWilliamson, Law et. al. present FlickerPrint, a computational analysis tool which can be used for measuring the interfacial tension and bending rigidity of soft fluctuating bodies, including biomolecular condensates, droplets or vesicles, from confocal microscopy images using flicker spectroscopy. This method is highly scalable so can be used to analyse the properties of whole populations of thousands of such soft bodies. 2 MotivationBiomolecular condensates play fundamental roles in sub-cellular organisation and it is well-known that the composition of condensates can affect their function. Measuring the conden-sates mechanical properties (for example, interfacial tension and bending rigidity) can aid the understanding of their biomolecular composition and cellular functions. However, measuring the properties of individual condensates under physiological conditions is very challenging and cum-bersome to scale to the population level using traditional methods. To overcome these issues, we have developed a software package to run flicker spectroscopy analysis of condensates at scale, to determine their interfacial tension and bending rigidity. At the same time, FlickerPrint can be harnessed to analyse other soft, fluctuating bodies such as lipid vesicles. 3 SummaryAccurate measurement of the mechanical properties of biomolecular condensates is an essential step in understanding their behaviour within cells. We present FlickerPrint, an open-source Python package to determine the interfacial tension and bending rigidity of thousands of biomolecular condensates using flicker spectroscopy by analysing their shape fluctuations in confocal microscopy images. We detail the workflow used by FlickerPrint to scale up these individual measurements to the population level and the computational requirements to run Flicker-Print. We provide examples of experiments in live cells and in vitro which are suitable for analysis with FlickerPrint as well as scenarios where the package cannot be used. Using these examples, we show that the results obtained from the analysis are robust to changes in the imaging setup, including frame rate. This implementation enables a step-change in the capability to measure two key properties of biomolecular condensates, the interfacial tension and bending rigidity. Moreover, the tools in FlickerPrint are also applicable for analysing other soft, fluctuating bodies, which we demonstrate here using vesicles.