Biological autoluminescence enables effective monitoring of yeast cell electroporation.

The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions.  To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. Saccharomyces cerevisiae culture was exposed to 8 pulses of 100 µs width with electric field strength magnitude 2-7 kV cm-1. To assess the sensitivity of our method in detecting yeast electroporation, we conducted a comparison with established methods including impedance measurements, propidium iodide uptake, cell growth assay, and fluorescence microscopy. Our results demonstrate that yeast electroporation can be instantaneously monitored during pulsing, making it highly suitable for industrial applications. Furthermore, the simplicity of our setup facilitates its integration into continuous liquid flow systems. Additionally, we have established quantitative indicators based on a thorough statistical analysis of the data that can be implemented through a dedicated machine interface, providing efficiency indicators for analysis.

Lab-on-chip microscope platform for electro-manipulation of a dense microtubules network.

Pulsed electric field (PEF) technology is promising for the manipulation of biomolecular components and has potential applications in biomedicine and bionanotechnology. Microtubules, nanoscopic tubular structures self-assembled from protein tubulin, serve as important components in basic cellular processes as well as in engineered biomolecular nanosystems. Recent studies in cell-based models have demonstrated that PEF affects the cytoskeleton, including microtubules. However, the direct effects of PEF on microtubules are not clear. In this work, we developed a lab-on-a-chip platform integrated with a total internal reflection fluorescence microscope system to elucidate the PEF effects on a microtubules network mimicking the cell-like density of microtubules. The designed platform enables the delivery of short (microsecond-scale), high-field-strength ([Formula: see text] 25 kV/cm) electric pulses far from the electrode/electrolyte interface. We showed that microsecond PEF is capable of overcoming the non-covalent microtubule bonding force to the substrate and translocating the microtubules. This microsecond PEF effect combined with macromolecular crowding led to aggregation of microtubules. Our results expand the toolbox of bioelectronics technologies and electromagnetic tools for the manipulation of biomolecular nanoscopic systems and contribute to the understanding of microsecond PEF effects on a microtubule cytoskeleton.

Biological autoluminescence as a noninvasive monitoring tool for chemical and physical modulation of oxidation in yeast cell culture.

Normal or excessive oxidative metabolism in organisms is essential in physiological and pathophysiological processes, respectively. Therefore, monitoring of biological oxidative processes induced by the chemical or physical stimuli is nowadays of extreme importance due to the environment overloaded with various physicochemical factors. Current techniques typically require the addition of chemical labels or light illumination, which perturb the samples to be analyzed. Moreover, the current techniques are very demanding in terms of sample preparation and equipment. To alleviate these limitations, we propose a label-free monitoring tool of oxidation based on biological autoluminescence (BAL). We demonstrate this tool on Saccharomyces cerevisiae cell culture. We showed that BAL can be used to monitor chemical perturbation of yeast due to Fenton reagents initiated oxidation-the BAL intensity changes with hydrogen peroxide concentration in a dose-dependent manner. Furthermore, we also showed that BAL reflects the effects of low-frequency magnetic field on the yeast cell culture, where we observed a disturbance of the BAL kinetics in the exposed vs. control case. Our results contribute to the development of novel techniques for label-free, real-time, noninvasive monitoring of oxidative processes and approaches for their modulation.

Geometrical constraint of sources in noninvasive localization of premature ventricular contractions.

The inverse problem of electrocardiography for localization of a premature ventricular contraction (PVC) origin was solved and compared for three types of the equivalent cardiac electrical generator: transmembrane voltages, epicardial potentials, and dipoles. Instead of regularization methods usually used for the ill-posed inverse problems an assumption of a single point source representative of the heart generator was applied to the solution as a geometrical constraint. Body surface potential maps were simulated from eight modeled origins of the PVC in the heart model. Then the maps were corrupted by additional Gaussian noise with the signal-to-noise ratio (SNR) from 20 to 10dB and used as the input of the inverse solution. The inverse solution was computed from the first 30ms of the ventricular depolarization. It was assumed that during this period only a small part of the heart volume is activated thus it can be represented by a single point electrical source. Generally, the localization error was more dependent on the PVC origin position than on the type of the used heart generator. The most stable localization error between the inversely found results and the true PVC origin was not larger than 20mm for PVC origins located in the left ventricular wall and on the right ventricular anterior side. For such cases, the localization was robust to the noise up to SNR of 10dB for all studied types of the cardiac generator. For SNR 10dB the results became unstable mainly for the PVC origins in the septum and posterior right ventricle for the dipolar heart generator and for epicardial potentials defined on the pericardium when the range of the localization error increased up to 50mm. When the results for different electrical heart generators were considered altogether, the mean radius of the cloud of results did not exceed 20mm and the localization error of the cloud center was smaller than that obtained for a particular type of the cardiac generator. Combination of results from different models of a single point cardiac electrical generator can provide better information for the preliminary noninvasive localization of PVC than the use of one type of the generator.

Noninvasive identification of two lesions with local repolarization changes using two dipoles in inverse solution simulation study.

BACKGROUND: The method for inverse localization and identification of two distinct simultaneous lesions with changed repolarization in the ventricular myocardium (two-vessel disease) is proposed and its robustness to errors in input data is tested in this simulation study. METHOD: The inverse solution was obtained from the difference between STT integral body surface potential map computed with repolarization changes and the STT integral map from normal activation. In a numerical model of ventricles 48 cases of two simultaneous lesions and 48 cases of a single lesion were modeled. The effect of the lesions was taken to be represented by two dipoles. The input data were disturbed by three types of added noise. Twenty three characteristics of every obtained inverse solution were defined and four of them were used as the features in discriminant analysis task distinguishing the correct inverse solutions identifying two lesions. RESULTS: The mean localization error for identified two lesions was 1.1±0.7cm. The sensitivity and specificity of quadratic discriminant analysis with cross-validation and feature selection was higher than 90%. CONCLUSIONS: The combination of the inverse solution with two dipoles and discriminant analysis allows the identification of two simultaneous lesions without a priori information about the number of lesions.