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

Abstract

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.

Fig. 1.

PoET method flowchart. The individual steps are explained in detail in the text.

Fig. 2.

GUI of PoET. (A) Main window exhibiting a processed video in the bottom panel. (B) Pore dynamics graph of the raw (dark pink solid circles) and smoothed data (blue open circles), suggested linear region (light blue area) and a linear fit (red line) to the slow pore closure stage used for pore edge tension calculation.

Fig. 3.

Comparison of manual and automated analysis. (A) Representative phase-contrast image sequence (360 frames per second) of a 10% CL GUV during application of an electroporating DC pulse (3 kV/cm, 150 µs). Scale bar corresponds to 50 µm. (B) Post-processed images obtained after analysis with PoET. Pore diameter, d, is indicated in each image. (C) Representative pore dynamics traces of 10% CL and 50% PG GUVs obtained manually by three analysts (blue, green and red circles) and automatically by PoET (black circles). (D) Edge tension values (individual measurements on different vesicles are displayed as circles; corresponding standard deviations are shown as error bars, open symbols show data from GUVs containing 10% CL and solid symbols relate to 50% PG) calculated from pore dynamics traces generated manually by three different analysts and automatically by PoET. Each arrow in (D) points to the edge tension value obtained from the matching color and style curve in (C).

Fig. 4.

Values of edge tension obtained for membranes made of pure PC (black squares), PC: CL (blue circles), PC: PI (green triangles), PC: PIP2 (magenta inverted triangles) and PC: PS (red diamonds). Each point (open symbols) corresponds to one measurement on a single GUV, and solid symbols show the mean values with standard deviation (vertical bars) for each condition studied.