Phase separation on PSMs

In this thesis, phase separation of PSMs was measured and analyzed in dependence of temperature and lipid mixture. The results of this study can be seen in chapter 5.2.

The lipid mixture varied in the Chol content from 0 to 50 mol % in a DOPC/SM 1:1 matrix. The measurement was performed with a CLSM within a temperature-control chamber.^[156] The prepared PSMs (chapter 3.2.3) on top of a silicon oxide (SiO_x) functionalized substrate were heated and cooled between 25 and 55^◦C. One measurement series was done on one membrane patch, either heated or cooled. The series consists of several images taken at certain temperatures. Different series were measured on one substrate with the same Chol concentration in the GUV solution.

The taken images are composed of two different channels. The channels display the intensity of the phase markers BODIPY-Chol and TexasRed-DHPE. There are two possibilities of phase separations located in the PSM within the measured phase diagram (figure 3.3). Firstly, the PSM phase-separated into the gel like (lβ)/ld phase at low temperature and, at high temperature, it is homogeneous in the ld phase.

Secondly, at low temperature, the phase separation is between thelo/ld phase. The phase separation was analyzed by eye for each series, comparing the images with the illustration in figure 3.3.

Figure 3.3.: The two different phase separations are illustrated with the different intensities of the two different fluorophores sulforhodamine-1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (TexasRed-DHPE) (red, upper line) and 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-Chol) (green, bottom lines) in the pore-spanning membranes (PSMs). The membrane is phase separated at temperature lower than the transition temperature (TM) in the gel like (lβ)/liquid disordered (ld) or liquid ordered (lo)/ld phase separation. At higher temperature than theTM the PSM is homogeneous in theld phase.

In the measurements and the illustration, the channel of TexasRed-DHPE is not influenced by the type of phase separation. It represents the ld phase, which is at lower temperature as the transition temperature (TM) always in the s-PSM and at higher temperature homogeneously distributed in the PSM. Therefore, this channel was used to identify theTM of each series.

In the first step, each fluorescence micrograph from all the temperature series and lipid compositions was separately analyzed to extract the intensity ratio of the f-PSM to the s-PSM. This was done with a custom designed Matlab-script. The analysis of the image sequence was performed in random order to avoid systematical errors from the user. The fluorescently micrograph analysis is schematically shown in scheme 3.13.

The two channels of the fluorescence micrograph show the fluorescence intensity of BODIPY-Chol and TexasRed-DHPE (scheme 3.13). To divide the fluorescence micrograph into the pore and rim regions, the channels were merged and smoothed with a median filter. The pore localization in the merged fluorescence micrograph was automated. Pores were not always identified as a circular object. Unidentified pores were extrapolated with the conditions of hexagonal pore orientation and equidistant spacing between the pores (scheme 3.13). As a control of the algorithm, the result was checked by visual judgment. If the calculated pore mask did not fit into the merged image (bad alignment), some pore centers had to be set manually, while the rest were extrapolated (scheme 3.13). The well aligned pore mask was used to separate the regions of the pores and the rim. The membrane areas (f-PSM and s-PSM) in both regions were found by threshold analysis. For that, an intensity histogram of the pore region or the rim region was created in order to have a decision guide for the threshold setting. Each pixel with an intensity above the threshold belongs to the membrane area, whereas each pixel with an intensity below the threshold belongs to the non membrane region, the so-called background. The generation of the f-PSM mask was shape constrained, because pores can only be completely spanned by membrane or not. This condition was realized with a filling parameter. If the amount of pixels which were higher than the threshold was greater than the filling parameter, the whole pore was included in the f-PSM mask, and if not the whole pore was included in the background mask. The mean intensity (I) of the TexasRed-DHPE channel was read out for the different areas by using these masks (f-PSM, Background, s-PSM).

Afterwards, the intensity ratio I_f-PSM

I_s-PSM = I_f-PSM −I_Background

I_s-PSM−I_Background. (3.24)

was calculated from the mean intensities of the TexasRed-DHPE channel.

The calculated intensity ratio of one series were plotted against the temperature (T) from the measured temperature ramp (figure 3.14). The TM is the tuning point of a sigmoidal curve,^[104] which was fit with

Scheme 3.13.: The process chart for the image intensity calculation. The two channels of the fluorescence image were merged to calculate the pore mask. The pore and rim regions were extracted and separately analyzed to get the masks of the membrane areas (freestanding pore-spanning membrane (f-PSM), solid supported pore-spanning membrane (s-PSM)). The non membrane areas were collected and assigned to the background. These masks were utilized to gain the mean fluorescent intensities of the specific regions from the TexasRed-DHPE fluorescence image (If-PSM,IBackground andIs-PSM).

I_f-PSM

I_s-PSM(T) =I_end+ I_start−I_end

1 +exp^T−T_dT^M. (3.25) The width of the transition (dT) describes the transition temperature range. The intensity ratio at low temperature is described through Istart and the intensity ratio at high temperature is I_end.

Scheme 3.14.: A schematically illustration of how to get the transition temperature (TM) of a temperature series form pore-spanning membranes (PSMs). The intensity ratio

I_f-PSM/I_s-PSMis plotted against the temperature. The turning point is theTM. The intensity ratio of the PSMs at three temperatures is illustrated above as fluorescence micrograph.

The measuredTM values of all samples with equal Chol concentration were aver-aged and plotted against the Chol concentration, as phase diagram.

4. Labeled globotriaosyl ceramides