PoET: automated approach for measuring pore edge tension in giant unilamellar vesicles.

Motivation: A reliable characterization of the membrane pore edge tension of single giant unilamellar vesicles (GUVs) requires the measurement of micrometer sized pores in hundreds to thousands of images. When manually performed, this procedure has shown to be extremely time-consuming and to generate inconsistent results among different users and imaging systems. A user-friendly software for such analysis allowing quick processing and generation of reproducible data had not yet been reported. Results: We have developed a software (PoET) for automatic pore edge tension measurements on GUVs. The required image processing steps and the characterization of the pore dynamics are performed automatically within the software and its use allowed for a 30-fold reduction in the analysis time. We demonstrate the applicability of the software by comparing the pore edge tension of GUVs of different membrane compositions and surface charges. The approach was applied to electroporated GUVs but is applicable to other means of pore formation. Availability and implementation: The complete software is implemented in Python and available for Windows at https://dx.doi.org/10.17617/3.7h. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

METROID: an automated method for robust quantification of subcellular fluorescence events at low SNR.

BACKGROUND: In cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community. RESULTS: In this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (- 10 dB) without apparent signal distortion. CONCLUSIONS: We demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at https://doi.org/10.6084/m9.figshare.11344046.v1 , and its code and datasets are available in GitHub at https://github.com/zoccoler/metroid .

Cardiomyocyte lethality by multidirectional stimuli.

Multidirectional defibrillation protocols have shown better efficiency than monodirectional; still, no testing was performed to assess cell lethality. We investigated lethality of multidirectional defibrillator-like shocks on isolated cardiomyocytes. Cells were isolated from adult male Wistar rats and plated into a perfusion chamber. Electrical field stimulation threshold (ET) was obtained, and cells were paced with suprathreshold bipolar electrical field (E) pulses. Either one monodirectional high-intensity electrical field (HEF) pulse aligned at 0° (group Mono0) or 60° (group Mono60) to cell major axis or a multidirectional sequence of three HEF pulses aligned at 0°, 60°, and 120° each was applied. If cell recovered from shock, pacing was resumed, and a higher amplitude HEF, proportional to ET, was applied. The sequence was repeated until cell death. Lethality curves were built by means of survival analysis from sub-lethal and lethal E. Non-linear fit was performed, and E values corresponding to 50% probability of lethality (E50) were compared. Multidirectional groups presented lethality curves similar to Mono0. Mono60 displayed the highest E50. The novel data endorse the idea of multidirectional stimuli being safer because their effects on lethality of individual cells were equal to a single monodirectional stimulus, while their defibrillatory threshold is lower. Graphical abstract Monodirectional and multidirectional lethality protocol comparison on isolated rat cardiomyocytes. The heart image is a derivative of "3D Heart in zBrush" ( https://vimeo.com/65568770 ) by Laloxl, used under CC BY 3.0 ( https://creativecommons.org/licenses/by/3.0/legalcode )/image extracted from original video.

Ventricular myocyte injury by high-intensity electric field: Effect of pulse duration.

Although high-intensity electric fields (HEF) application is currently the only effective therapy available to terminate ventricular fibrillation, it may cause injury to cardiac cells. In this study we determined the relation between HEF pulse length and cardiomyocyte lethal injury. We obtained lethality curves by survival analysis, which were used to determine the value of HEF necessary to kill 50% of cells (E50) and plotted a strength-duration (SxD) curve for lethality with 10 different durations: 0.1, 0.2, 0.5, 1, 3, 5, 10, 20, 35 and 70 ms. For the same durations we also obtained an SxD curve for excitation and established an indicator for stimulatory safeness (stimulation safety factor - SSF) as the ratio between the SxD curve for lethality and one for excitation. We found that the lower the pulse duration, the higher the HEF intensity required to cell death. Contrary to expectations, the highest SSF value does not correspond to the lowest pulse duration but to the one of 0.5 ms. As defibrillation threshold has been described as duration-dependent, our results imply that the use of shorter stimulus duration - instead of the one typically used in the clinic (10 ms) - might increase defibrillation safeness.