Marian Smoluchowski
Institute of Physics

Supervisor: prof. Ewa Stępień

Defence year: 2023

Investigating the ortho-positronium (o-Ps) lifetime in biological materials holds promise for enhancing our understanding of biological processes and pathologies. This master?s thesis aimed to develop experimental conditions for the measurement of the o-Ps lifetime in extracellular vesicles (EVs) isolated using the low-pressure filtration method. EVs in question were derived from a largescale 2D human pancreatic beta cell culture (1.1B4 cell line) maintained under normoglycemic conditions. In order to check the effectiveness of the chosen method of isolation, environmental scanning electron microscopy (ESEM) images of the dialysis membrane were taken, which allowed to determine the size of its pores. Fourier transform infrared spectroscopy (FTIR) was employed to characterise the chemical composition of EVs. The qNano device, which operates on the principle of tunable resistive pulse sensing (TRPS), was used to determine the size distribution and concentration of EVs. Transmission electron microscopy (TEM) was employed to visualise the morphological features of EVs and validate their structural integrity. Finally, positron annihilation lifetime spectroscopy (PALS) was used to investigate the o-Ps lifetime in the EV sample. In addition, the PALS technique was also used to study the lifetime of o-Ps in pancreatic beta cells from which the vesicles were derived, and in PBS buffer in which they were suspended after the isolation. The o-Ps lifetime and intensity for the sample containing EVs measured at 22°C were 1.87 ns and 15.4%. The same sample investigated at 34.2 °C gave results of 1.83 ns and 15.0%, respectively. These results were not significantly different from the results of the PBS measurement. This study allowed to test several technical solutions to measure the positronium lifetime in EV samples. It underscores the need for continued efforts in exploring EVs with progressive methods such as PALS, as they hold immense potential, for example as carriers of biological cargo or biomarkers.

Supervisor: prof. Ewa Stępień

Defence year: 2021

The phenomenon of positronium formation in biological material brings new possibilities formedical diagnostics. This thesis presents a method for determining mean positronium lifetme in microvesicles isolated from the culture of beta-pancreatic cells using low-vacuum filtration technique combined with the ultracentrifugation. The concentration of microvesicles in the prepared samples was tested with qNano particle analyzer, the operation of which is based on the TRPS (Tunable Resistive Pulse Sensing) technology. Before starting the actual measurements, Positron Annihilation Lifetime Spectrometer was adjusted accordingly, e.g. by equipping it with a thermostat which allows to precisely control the temperature in the system. A series of measurements was performed to check the temperature stability and a calibration curve was obtained, on the basis of which it is possible to accurately determine the temperature of the sample during the measurement. The positron source was additionally secured against leakage, which may occur when testing with liquid samples. Parafilm was used for this purpose, the positronium lifetime in it changes with temperature, what was taken into account in data analysis. The positronium lifetime in microvesicles cultured under normoglycemic and hyperglycemic conditions was investigated. The values were 1.80 ns and 1.77 ns respectively, thus not significantly different from the lifetime determined in the PBS buffer in which microvesicles were suspended (g = 1.80 ns). On the basis of the obtained results, it can be concluded that to conduct research with microvesicles using the presented method, it is necessary that the samples have a higher concentration of vesicles.