Quantitative analysis of fluorescence changes upon membrane permeabilization

It was necessary to restrict the image acquisition to time-resolved z-lines (cross sections) of the sample because of long acquisition times of several minutes per image in the confocal laser scanning microscope setup. It was assumed that the observations in a cross section of the patch are representative for the whole patch. Additionally, the state of the membrane patches before and after AMP administration was documented with single 2D images in xy plane.

The acquisition of a single z-line required the development of a way to compensate for focus drift.

3.3.5.1 Visual representation and analysis of fluorescence intensity changes

The time course of dye inflow was observed as change in fluorescence intensity [IROI(t)] in a region of interest (ROI) under the membrane patch. Additionally, the intensity of a ROI in non-sealed cavities [Iref(t)] was set as a reference for complete pore filling (see Fig. 3.12). The relative fluorescence intensity [Irel(t)] as a function of time t was calculated by normalizing IROI(t) by the fluorescence intensity at t = 0 (I0) and standardizing against Iref(t), a Boltzmann function was fitted to the data to analyze the time course of the resulting sigmoidal curves, as described in 2.4.4.

Fig. 3.12 illustrates with a representative data set how center time (t1/2), tangent slope (τ), and maximum fluorescence intensity (IMAX) as values for the time point of 50 % pore filling, filling speed and maximum filling degree were obtained on a representative dataset (see 2.4.4.). For the acquisition of the dataset, pore-spanning membranes were prepared as described in 3.3.1. 6.6 µM magainin-2 was added, and fluorescence changes were analyzed on z-line images.

59 Especially in the initial phases after AMP addition, dye inflow occurred in the form of filling of individual cavities, seen as vertical green spikes below the membrane. The resolution limit of optical microscopy did not allow for “spike counting”, that is, the quantification of individually filling cavities over time. For this reason, the overall cavity fluorescence was measured.

For this purpose, a MATLAB script was used that allowed to quantify relative fluorescence changes in the cavities. It was also necessary to compensate for z-drift of the image focus during the time course of the experiments. The script was developed by Dr. Ingo P. Mey, who generously provided it for the use in this work.

In short, the script uses the data obtained from z-lines of time-lapse experiments to identify the phase boundary between AAO substrate and supernatant buffer. This phase boundary was aligned in all individual images of the dataset. The area below the membrane patch was marked by a region of interest (ROI, Fig. 3.12 top, yellow box), and time-resolved

Fig. 3.12: Measurement of membrane changes over time during AMP administration. (A) Overlay of exemplary fluorescence images showing z-line profiles of pore-spanning Texas Red DHPE labeled POPC bilayers (red) on AAO substrate and 1 mM pyranine (green) in bulk phase at the given time points. Images show the lipid bilayer state before addition of 6.6 µM magainin-2 (t = 0), at the time where the fluorescence intensity reaches half its maximum value (half time t1/2), and in the equilibrium state, where the maximum fluorescence intensity in the pores is reached (IMAX). The large yellow box represents the region of interest (ROI) used for the time-resolved quantitative analysis of the pyranine dye entrance by measuring fluorescence intensity, IROI. The turquoise box presents the reference ROI, where Iref is measured. Upon addition of magainin-2, pyranine fluorescence (green) becomes visible in the AAO cavities that were prior sealed by the pore-spanning membrane (red). Scale bar 20 µm. (B) Graphic representation of the time course of measured Irel (pink) as a function of time, with Boltzmann fit (black). The graphic representation allows for determination of half time t1/2, tangent slope τ of the sigmoidal curve at the inflection point, and maximum intensity IMAX at the equilibrium state at the end of the experiment.

A

B

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quantitative fluorescence values were obtained yielding the ROI intensity IROI over time. In a reference measurement (ref, Fig. 3.12 top, turquoise box), Iref was obtained and used for normalization of IROI.

The changes of the obtained fluorescence intensity values depend on AMP concentration, and can be used to determine the speed and degree of filling at the end point of the experiment.

This information allows for conclusions on the modes of membrane permeabilization of different agents. The MATLAB script as well as a visual example of the dataset alignment are available in the appendix.

With these measures, it was possible to visualize and quantitatively compare kinetics of dye inflow under different membrane permeabilization conditions.

In a control experiment, it will be shown that the quantitation and graphic representation could be successfully implemented.

3.3.5.2 Proof of principle: Quantitation of lipid bilayer lysis with Triton X-100

To obtain a reference measurement on complete lysis of the lipid bilayer on porous substrate support, the membrane patches were treated with 0.5 µM Triton X-100. This detergent efficiently solubilizes lipid membranes through micellation of the lipids.

For this, a membrane preparation was made as described in 3.3.1, where POPC GUVs labeled with 0.5 mol% Texas Red DHPE were spread onto closed AAO pores. Triton X-100 in a final concentration of 0.5 µM was added to the supernatant buffer. Three water-soluble dyes were added to the bulk volume and analyzed to monitor membrane permeability: FITC-labeled dextrans (40 kDa and 70 kDa) at a final concentration of 5 µM, as well as pyranine at a final concentration of 1 mM.

61 Fig. 3.13 depicts the measured fluorescence intensities of the three dyes before and after Triton X-100 addition. An immediate increase in fluorescence inside the substrate pores was observed upon Triton X-100 addition. This increase was observed independently of the used dye. These measurements provided the values for the maximum fluorescence values with these experiment parameters. The obtained relative fluorescence values were standardized to 0 (initial fluorescence) and 1 (maximum fluorescence). They will be used later in this work for comparison where appropriate.

To sum up, a routine was successfully established to visualize and analyze changes in fluorescence intensities in the microscopy experiments of lipid bilayers on AAO support.

3.4 Investigation of AMP permeabilization modes using kinetic fluorescence analyses