Combination of topographic AFM imaging and STED

The observed globular structures on the membrane sheets might hinder the interaction between the nanobodies on the cantilever and their binding partner on the membrane. The distribution of syntaxin-1 might, furthermore, be related to these structures. In that sense, a

hindrance of the interaction will be expected if the syntaxin-1 clusters observed in STED microscopy (Figure 4.4) colocalise with the elevations measured by AFM. To test this hypothesis, membrane sheets from PC12-WT-2 cells were stained for syntaxin-1, imaged with STED microscopy and subject to AFM height imaging afterwards (N = 3 sheets from a single preparation). Figure 4.34 shows an overlay of obtained AFM and STED images.

Figure 4.34: Overlay of an AFM height and a STED image of syntaxin-1 of a PC12 membrane sheet. The greyscale image shows the topography of a membrane sheet derived from a PC12-WT-2 cell. Blue pixels correspond to erroneous values or heights above 1 µm. The syntaxin-1 label measured by STED microscopy is shown in green.

Scale bar: 5 µm.

For the membrane sheet shown in Figure 4.34 the correlation analysis between the height and the binarised fluorescence image of the syntaxin-1 label was performed inside a quadratic ROI of 9.6×9.6 µm² in a central region. In Figure 4.35 the cumulative probability of the height of all pixels inside that ROI (black), of the ones colocalising with syntaxin-1 in the binarised STED image (red) and for control purposes those obtained from rotating the STED image stepwise by 90° (cyan and blue, see caption of Figure 4.35) are shown. Generally, the distribution of height values of the regions which are, according to the STED image, occupied by syntaxin-1 (red line in Figure 4.35) resembles that of all pixels inside the ROI (black line in Figure 4.35). The slight deviation from control curves (blue and cyan lines in Figure 4.35) at around 0.4 µm is a unique feature of that membrane sheet and not found for the other two investigated membrane sheets. Consequently, the heights of the membrane sheets in regions with present signal from the syntaxin-1 label do not deviate from the overall height distribution. Thus, it can be concluded that the apparent distribution of syntaxin-1 is most probably not related to the topographical features of the membrane sheet.

4.1 Heterogeneity and Clustering in PC12 Membrane Sheets

Figure 4.35: Quantification of the topography of regions of the membrane sheet occupied by syntaxin-1 compared with the whole ROI. The cumulative probability is plotted against the height of the membrane sheet measured by AFM. The back line corresponds to height values of all pixels inside a ROI on the membrane sheet shown in Figure 4.34. The red line represents height values of pixels inside the ROI which colocalise with syntaxin-1. For control purposes the cyan lines correspond to pixels detected by rotating the binary mask used to identify regions occupied by syntaxin-1 by 90°, 180° and 270°. The blue line is the cumulative probability of the height values of all three cyan control curves.

4.1.11 Topography of membrane sheets derived from primary neurons

PC12 cells have the advantage of relative time efficient procedures in cell culture. However, as pronounced elevations on membrane sheets derived from PC12 cells have been revealed by AFM height imaging as shown in section 4.1.9, it was sought for an alternative system for MR-AFM imaging of syntaxin-1 for future experiments. To this end, the topography of membrane sheets derived from primary rat hippocampal neurons was imaged by FD-AFM, as shown for a representative example in Figure 4.36 (N = 7 images from three sheets).

The image recorded at the border of the membrane sheet (Figure 4.36 a) shows the membrane sheet (middle grey) distinguishable from the substrate (dark grey) with present elevated structures (light grey). The image recorded closer to the centre (Figure 4.36 b) details the presence of structures which are quite similar to the ones found on PC12 cell membrane sheets.

Figure 4.36: Topographical investigation of membrane sheets derived from primary neurons. In a a FD-AFM height image of a region at the border of a membrane sheet is shown, the one in b is recorded exclusively on the membrane of the same sheet. Blue pixels denote erroneous values and in a heights above 300 nm, additionally. The colour scale holds for both images. Scale bars: 1 µm. In c histograms of the height values are shown. The magenta coloured histogram corresponds to the image shown in a and the red one to that shown in b. For comparison, the histogram of the PC12-WT-1 membrane sheet already shown in Figure 4.32 is added in blue. The histograms are cut at h = 200 nm, thereby not showing height values accounting for 1.7 % and 0.06 % of all values for the histogram shown in magenta and red, respectively.

At first glance, these structures do not seem to be as dense as in PC12 cell membrane sheets, suggesting the possibility to perform MR-AFM measurements in between the elevations. However, a more detailed analysis of the distributions of height values shown in Figure 4.36 c indicates a good agreement in the distributions of heights in the images shown for primary neurons (red histogram) and for PC12-WT-1 cells (blue histogram). The larger offset in the magenta coloured histogram, which corresponds to the image measured at the border of the membrane sheet (Figure 4.36 a), is caused by the fact that the substrate is set to a height of zero instead of the lowest height on the membrane sheet, thereby shifting the height distribution about 10 nm towards larger height values as compared to the histogram shown in red. This shift can therefore serve as an estimate of a hypothetical flat membrane without the additional elevations.