Atomic force microscopy

Binning et al. first introduced the atomic force microscopy (AFM) technique in 1985.⁸⁰ It enabled scanning of non-conductive probes under physiological conditions with a high lateral (1.0 nm) and axial (0.1 nm) resolution.⁸¹ Hence it is well suitable for the analysis of biological systems, like e.g. receptor-ligand interactions.⁸² In this work AFM was used to analyze surface topographies of solid-supported membranes and ENTH clusters.

Principle of an AFM

A cantilever with a sharp tip is moved over a sample by x-y piezo actuators. Further-more, a z-piezo actuator can approach the cantilever towards the sample until attrac-tive or repulsive interactions of the sample and the cantilever lead to the deflection of it. The deflection is detected by a laser beam, which is reflected by the cantilever surface as well as by a mirror and then directed to a four quadrant diode. Lateral or vertical deflection results in the shift of the laser beam on the diode. Hence the deflec-tion of the cantilever against the sample posideflec-tion allows to get a topographic image of the sample surface. The setup of an AFM is illustrated in Figure 3.10.

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Figure 3.10: Schematic illustration of an AFM setup. The piezos move the cantilever on the surface. A light beam, which is directed to the cantilever, is reflected and directed to the four quadrant photodi-ode, detecting the deflection of the cantilever.

In AFM different imaging modes can be applied. In this work the contact mode was used, where the tip is in close contact to the sample. The contact mode can be realized in constant height or constant force. In case of constant force a topographic image can be illustrated due to the piezo element regulation. This mode appeared to be best suited for imaging ENTH clusters on solid-supported membranes.

Besides topographical information, AFM is utilized for the determination of mechan-ical properties of samples by force-distance curves. This method is often used to measure the elastic characteristics of biological systems.⁸³ Thereby, the force can be obtained by the vertical cantilever defection Zc and the spring constant of the cantile-ver (k) by Hooke’s law (equation (3.19)).⁸⁴

𝐹 = 𝑘 ∙ 𝑍_c (3.19)

For force-distance curves the position of the piezo Zp and the cantilever deflection Zc

are converted into force F and distance D (Figure 3.11 B). Applying a fit to the com-pliance region yielded the slope, which is equal to the conversion factor of the canti-lever defection and the detector signal. The tip-sample distance can be obtained by equation (3.20).⁸⁵

𝐷 = 𝑍_p+ 𝑍_c (3.20)

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Force-distance curves were detected at single sample positions and predefined forces of 6-8 nN to obtain breakthrough forces which provides the membrane thicknesses as they are correlated (Figure 3.11).

Figure 3.11: Schematic illustration of a signal versus piezo position (A) and a force-distance curve (B).

During approach (blue) no interactions of cantilever and sample occur (I). Due to attractive forces the cantilever gets in contact with the sample and the measured force suddenly decreases (II). Further approach leads to increase of the force (IV). At high predefined forces the cantilever can break through the lipid bilayer resulting in a second drop of the force (III). Until the retraction energy exceeds the adhesion energy the cantilever stays in contact to the surface during retraction (red, V). At the end the baseline is reached again as no contact of cantilever and sample is present.

By the approach of the cantilever the force suddenly decreases due to attractive elec-trostatic or van-der-Waals interactions. As the cantilever is in contact further ap-proach results in the increase of F until a predefined value. At high predefined forces a small drop in the approach force-distance curve can appear (cantilever tip breaks through the bilayer), enabling the determination of the membrane thickness. In the force-distance plot the membrane thickness is the distance between the decrease of F and the point where F begins to rise again (cf. Figure 3.11 B).⁸⁶ Then retraction of the cantilever leads to the decrease of F. An even negative force value can be reached when adhesion causes cantilever bending. With further retraction the cantilever loses the contact as the retraction force exceeds the adhesion force.

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For the supported lipid bilayers (SLBs) previously described FRAP and CLSM experi-ments (cf. chapter 3.3.3.1) were done to verify the mobility of the membranes. Meas-urements were performed using a JPK Nanowizard 4 (JPK Instruments, Berlin, GER).

After fixing the cantilever to a glass holder and implementing both into the AFM head, the laser was directed to the tip of the cantilever to get a maximal signal. The exact spring constant of the cantilever was determined by measuring the thermal noise spectrum.⁸⁷

As all settings were adjusted, micrographs of SLBs were taken to analyze the surface topography before and after protein adsorption. SLBs were prepared as described in chapter 3.2.2. Measurements were done in contact mode using BL-AC40TS-C2 canti-levers (BioLever mini, f = 85.4-139.1 kHz, k =0.03-0.12 N/m, Olympus). First of all break-through experiments were performed to measure the membrane thickness as it can be derived from the break-through force. Besides FRAP experiments, this en-sured that a bilayer with a thickness of about 4 nm was formed, which is typical for a bilayer.⁸⁸Next, an area of 10 x 10 µm² of the SLBs was imaged. After incubation with 1 µM ENTH or ENTH R114A mutant for 2h at RT, the surface was scanned again.

Roughnesses (root-mean-square, rms) were determined with the integrated JPK Data Processing software.

The protein height and occupancy were analyzed using a MatLab script written by Dr.

Ingo Mey (Georg-August Universität Göttingen). To detect the membrane and the pro-tein a threshold was set. Propro-tein adsorption was identified by a 2D peak detection function, which marked the local maxima. A histogram out of the maxima was created and fitting a normal distribution yielded the protein height.

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