| Sumario: | In this article, we propose a method for automated tracking and analysis of vesicle contours in video sequences acquired by phase contrast microscopy. The contour is determined in each frame of the selected video sequence by detecting the transition between the interior and exterior of the vesicle that is reflected in the image intensity gradients. The resulting contour points are represented in the polar coordinate system, i.e., with uniform angular sampling and with coordinates that originate from the vesicle center of mass, enabling the analysis of the vesicle shape and its membrane fluctuations. By analyzing artificial images with known ground-truth contours, the accuracy and precision of the proposed method was estimated to be 34.1 and 26.9 nm for image signal-to-noise ratio of 23 dB and pixel size of 35 nm, respectively. The proposed method was evaluated on quasi-spherical vesicles made up of different proportions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol and exposed to different temperatures. The results show that the method is robust and efficient in terms of speed and quantitative description of vesicle fluctuations. The magnitude of vesicle membrane fluctuations increased with temperature, while the bending rigidity of the membrane was increasing for temperatures up to 20 °C and decreasing for higher temperatures irrespective of the vesicle molecular structure.
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