Automated tracking and analysis of phospholipid vesicle contours in phase contrast microscopy images

Abstract

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.

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F 1 F 0 -ATP synthase

“…Experiments on membrane fluctuations were made at 22 °C. The GUV equatorial positions were digitally segmented using a custom-made algorithm that combines accurate instantaneous positioning of the membrane contour with respect to the GUV center ( 55 ) and optimal contour imaging with respect to the background noise ( 11 ).…”

A gradient-based, GPU-accelerated, high-precision contour-segmentation algorithm with application to cell membrane fluctuation spectroscopy

“…The performance of contour-segmentation algorithms for tracking membrane fluctuations from video-microscopy images has always played a crucial role in the advance of this technique by enabling a more precise determination of the cell contour-position [13]. Accordingly, various segmentation methods have been proposed with contrast imaging [7,13–21], which were accompanied by technological advances in digital image processing and computational power. The contour-segmentation algorithm we present here harnesses the computational power of general purpose graphics processing units (GPGPU) having recently become available to significantly improve on current segmentation methods in the sub-pixel performance of nanometer resolution.…”

“…Early segmentation algorithms used the intensity maximum of the halo to determine the location of the cell-contour [14,16,19,27]. However, it was later pointed out by Döbereiner [18] and Pécréaux [13] that the location of the interface is actually placed at the maximal gradient of the halo intensity [28] and this has become the preferred method for the membrane localization [13,16–18,20,21]. Pécréaux and cols.…”

“…In the Pécréaux’ algorithm segmentation is performed sequentially along the contour using a number of different conditions to determine the segmentation direction. More recently, new segmentation methods have been proposed to locate the contour positions by directly determining the maximum of the intensity gradient [20,21]. In particular, Usenik et al [21] used interpolation to calculate the image intensity along radial directions starting from the center of the contour.…”

Suppression of membrane vesiculation as anticoagulant and anti-metastatic mechanism. Role of stability of narrow necks

“…The vesicle outlines were determined from the corrected images (Sevšek and Gomišček 2004;Usenik et al 2011). The images were binarized and slightly smoothened if necessary.…”