TITLE: 4 Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA
AUTHORS: Philip J. Haynes, Kavit H. S. Main, Alice Pyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 4 of the "Atomic Force Microscopy of DNA and DNA-Protein Interactions" collection of protocols.</div><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and it’s interactions with proteins.</span></div></div>

[STEPS]
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Once the tip has reached the surface, minimize the setpoint to the point at which the maximum force barely exceeds the force noise (~70 pN).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Optimise Sync Distance New such that the marker (a small diamond on the force curve, see Fig. 3c) aligns with the peak of the force curve. Set the Sync Distance QNM equal to this for imaging and check that the Force-Z curve is ‘folded’ about the point of maximum force (see Note 21).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Reduce the total length of the force curve (z length or ramp) to ≤10 nm, reducing hydrodynamic drag and maximising sampling near to the sample surface (see Note 22).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Adjust the Lift Height, such that the baseline of the force curve is flat (Fig. 3c). Autoconfig will also reset the background subtraction, but may perturb the sample.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Begin scanning an area of ~500x500 nm2.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Locate a DNA molecule of interest.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Adjust the Feedback Gain (if appropriate) to ensure the molecule is adequately tracked (see Note 29).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
If the molecule cannot be tracked (see Note 23), increase the force to allow tracking of the molecule. This will also require readjustment of the gains.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Ensure that the molecule being tracked is stable under imaging by verifying that it does not significantly shift between subsequent scan lines.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Reduce the Scan Size by zooming in on the DNA molecule of interest.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Increase the number of pixels per line to obtain ~0.5 nm per pixel (e.g., 256 pixels per line for a 120 nm scan).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Reduce the applied force by reducing the PeakForce Setpoint and re-adjust the gains if required (see Note 24).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Lateral drift or creep may be visible as the objects appearing to move across the image between subsequent scans (see Note 25). Under such conditions, higher scan speeds, or allowing the microscope to further equilibrate may improve resolution.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Align the molecule to the direction along which the scan lines are recorded (the fast scan direction) for highest resolution.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
If the part of the force curve above the baseline appears sinusoidal, as opposed to flat. The background will need to be recalculated by re-adjusting the Lift Height (see Note 26).
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Optimize the applied force and gains by increasing and decreasing the force in the range where the DNA molecule is not overly compressed (i.e., the measured height of the DNA should be ~20% of its known 2 nm diameter, see Fig. 4) to maximize resolution.
?. [Optimizing AFM Imaging in PFT for High Resolution AFM Imaging on DNA]
Other imaging parameters will vary depending on the antilever. Parameters when using an Fast-Scan D are outlined in Table 1.  AB1PFT ParametersTypical value2Scan Size [nm]120 - 2503Pixel density [pixels/line]256 - 5124Line rate [Hz]3-55Imaging PeakForce Setpoint [pN]~ 70 *6PF acquisition Frequency, see Note 19 [kHz]87LP Deflection Bandwidth [kHz]208Sync Distance, see Notes 19 and 21 [μs]70 / 209PeakForce Amplitude, Note 22 [nm]5-10 *10Lift Height, see Note 26 [nm]5-711Z Range (Z Limit if using MultiMode® 8) [nm]500-100012Deflection Limit [V]⋜12.24
AB1PFT ParametersTypical value2Scan Size [nm]120 - 2503Pixel density [pixels/line]256 - 5124Line rate [Hz]3-55Imaging PeakForce Setpoint [pN]~ 70 *6PF acquisition Frequency, see Note 19 [kHz]87LP Deflection Bandwidth [kHz]208Sync Distance, see Notes 19 and 21 [μs]70 / 209PeakForce Amplitude, Note 22 [nm]5-10 *10Lift Height, see Note 26 [nm]5-711Z Range (Z Limit if using MultiMode® 8) [nm]500-100012Deflection Limit [V]⋜12.24