DIRECTORA DE LA OFICINA MUNICIPAL DE LA MUJER

To optimise our imaging conditions in ‘peak force tapping’ mode, we can control the load force applied to the sample as described in 2.2.5. Peak force tapping mode essen- tially performs a force curve at every pixel position on the sample surface. It then uses a continuous feedback loop to detect the sample position and apply a pre-determined force defined by the user, referenced with respect to the force baseline that is deter- mined at every force curve as well. The resulting precise control of the load force is an advantage over the traditional tapping mode, in which the drift in resonance peaks within the fluid cell, caused by the mechanical resonances within the cell in addition to the cantilever, leads to uncertainties in the applied force. Figure 4.4 shows the effect of increasing the force on the imaging of DNA, both in the larger topographic features and in the resolution.

Figure 4.4: A DNA plasmid imaged at peak load forces of (A) 40, (B) 70, and (C) 190 pN, respectively, with the major and minor grooves of the DNA double helix visualized at higher magnification (insets). Colour scales (see figure 4.3 for scale bar): 3 nm (for low magnification); 2 nm (for the insets). (D) Height profiles measured across the DNA, as marked on the inset of A by a dashed line, for different peak forces. (E) Measured height along the same section across the molecule (as D), as a function of peak force.

Figures 4.4A-C show how the molecule is compressed under increasing force. The colour scale is the same for each image (3 nm). However, the DNA appears darker

(lower) as the force is increased. This increase of force also disrupts the structure, as shown by the arrow in figure 4.4C, showing a movement in the plasmid for imaging at a peak force of 200 pN. In addition to the overall compression, we can also examine the effect that increased force has on imaging the secondary structure of the molecule. The insets in 4.4A-C show smaller scans of the molecule, in the area highlighted by a white rectangle in figure 4.4A. These smaller scans show the secondary structure of the molecule as clear lines perpendicular to the direction of imaging.

Figure 4.4A is taken at a setpoint of 40 pN, a setpoint close to the minimum possible force which can be applied in peak force tapping to stay above the noise threshold. At this force, corrugation corresponding to the double helix is visible in the inset, and the height is calculated accurately at ∼2 nm (see figure 4.4D for a line profile showing this). However when we look at the image we see some ‘parachuting’, where the tip does not follow the contours of the sample well when scanning strands perpendicular to the fast scan direction. This occurs when the setpoint is too low for the feedback to accurately track the sample. At 70 pN, as in figure 4.4B, we record around 20% compression of the structure, and the feedback accurately traces the sample surface, as verified, e.g., by comparing the left-to-right (‘trace’) and right-to-left (‘retrace’) scan lines. In addition, the corrugation is more visible across the entirety of the plasmid in the large image. In the inset, the corrugation seems to be better resolved than in the large image. At higher forces of 190 pN, we see little evidence of corrugation, in either the main or inset images in figure 4.4C. There is significant compression, and areas where the plasmid appears very thin as it is moved by the force exerted by the tip. The compression visible in figures 4.4A-C is shown in figure 4.4D. The heights were measured for each inset along the line marked in figure 4.4A, with a 0.5 nm width on the line profile to reduce any errors. At 40 pN, the minimum peak force, the measured height of the DNA (1.9 _{± 0.2 nm) agrees with the diameter of B-DNA. At about 70} pN there is 20% compression of the molecule, this is when the corrugation is most pronounced. At up to 200 pN, the corrugation is less clear and the measured heights reduce to <1.5 nm, similar to most earlier tapping mode AFM experiments in liquid. In figure 4.4E we plot the measured heights for a number of plasmids, measured in the same manner as for figure 4.4D. This gives rise to a clear trend in compression as a function of the applied force. This trend could be used in traditional tapping mode experiments in fluid, with the DNA molecule acting as a ‘force gauge’ to estimate the applied force during imaging.

Figure 4.5 shows that this effect is reversible for forces of up to 200 pN. The imaging was performed by increasing the force to 200 pN and subsequently reducing this back to the minimum force of _{∼30 pN. The experiments were stopped at a maximum force} of 200 pN as tip damage occurs at forces higher than this. The evidence of corrugation after imaging at 200 pN implies that at this force we can retain the sharpness of the tip.

Figure 4.5: Repeatability of DNA height measurements in the AFM topography. (A) Measured height of the DNA plasmid on repeated ramps of the peak force. (B) AFM topography recorded at a peak force of 65 pN, after the two force ramps of (A), showing that the plasmid is still intact. Colour scale (see figure 4.3 for scale bar): 3.7 nm. The data sets taken match well as expected, following the same trend as figure 4.4D. The black data points here are determined from high-magnification images for peak forces increasing from 40 to 190 pN, the blue data points were collected from lower magnification images and thus have larger error bars. This shows the repeatability of these measurements as a technique for verifying applied force in AFM experiments conducted in fluid.