The first biological molecule observed using atomic force microscopy (AFM) was DNA. For the study DNA structure, dynamics, topology and interaction with proteins, the primary technique for imaging continues to be AFM.
Using the exclusive PeakForce Tapping® technology from Bruker, high-resolution imaging of the DNA double helix can be performed at imaging forces that can be quantified, without the need for restrictive AFM designs or specialized probes.
The Introduction of AFM into Biological Research
AFM has been extensively used for biological research from the time the TappingMode™ was introduced in the early 90s. In TappingMode, the oscillation of the probe takes place at its fundamental resonance frequency and the tip’s vertical position is adjusted continuously to maintain a constant oscillation amplitude as the probe scans across the surface.
TappingMode offers several benefits while studying biological sample structure, however critics believe that in comparison to contact mode imaging, it offers low-resolution images of biomolecules.
PeakForce Tapping Mode for Routine High-Resolution Imaging of Biomolecules
The PeakForce Tapping AFM imaging mode was launched by Bruker in 2010 and within a short period, it has been extensively used for studying biomolecules. The tip-sample distance is modulated in a sinusoidal motion at amplitudes normally below 100nm and at frequencies of 1 or 2kHz in PeakForce Tapping mode.
As shown in Figure 1a, when the AFM probe is brought into contact with the surface of the sample, the tip-sample interaction is controlled by maintaining the peak force or the maximum force between the tip and the sample constant. Considering the probe movement in terms of Z position, a force curve is performed at every pixel position on the sample surface (Figure 1b).
The benefit of PeakForce Tapping is that it uses a constant feedback loop for adjusting the relative tip-sample position. Instead of linear ramping, PeakForce Tapping employs sinusoidal ramping so that as the tip approaches the surface, the tip velocity approaches zero. These features ensure accurate and direct control of the tip-sample interaction force, enabling imaging in fluids at forces of 100pN or less.
Hence, both the sample and the AFM probe are protected from damage. Furthermore, imaging in PeakForce Tapping is more rapid when compared to other force-distance curve-based imaging modes. Thousands of force curves per second are possible with PeakForce Tapping as it operates at very high frequencies (1-2kHz).
Figure 1. In PeakForce Tapping Mode the AFM probe is modulated at low frequency (1-2 kHz). (A) As the probe is brought into contact with the surface, the feedback signal is the maximum or “peak” force applied to the surface. (B) If the motion of the probe is considered in terms of Z position, one is essentially performing a force curve at every position of the sample surface.
PeakForce Tapping technology has also enabled the self-optimizing ScanAsyst imaging mode. With ScanAsyst, setpoint drift that normally occurs in other AFM operating modes because of cantilever deflection drift and/or resonance peak shifting is prevented, due to optimization of the imaging setpoint.
Since the imaging force at the point of each tip-sample interaction is auto-optimized, high-resolution images can be obtained with the PeakForce Tapping technology. Along with auto-optimization of other parameters in ScanAsyst mode, for instance gain and scan rate, PeakForce Tapping enables rapid and reliable data irrespective of user skill levels.
By imaging single virus capsids, it is possible to illustrate the performance of PeakForce Tapping. In previous studies involving the use of the TappingMode, the arrangement of the virus particles was performed in a 2D crystal structure.
This 2D array offers mechanical stability to the virus particles so that they are not damaged when the AFM probe is applied. Using “jumping mode,” AFM high-resolution images can be obtained, considering the fact that virus capsids are very delicate.
As in PeakForce Tapping, in jumping mode, discrete force curves are created along the fast scan axis with topography data derived from the force curves. However, for every individual force curve in PeakForce Tapping, background artifacts, due to viscous drag caused by cantilever motion in fluid, are subtracted by the real-time feedback loop. By removing the background, the sensitivity of peak force detection is improved and lower imaging forces can be deployed.
A PeakForce Tapping image of a single herpes simplex virus is shown in Figure 2. Protein molecules are arranged as 3D subunits on the virus capsid’s surface and are called capsomeres. These are now very clearly visible. It is significant that the imaging of these virus particles was performed as isolated and individual particles without lateral stabilization.
Figure 2. 3D topography image of a single herpes simplex virus obtained in ScanAsyst mode in buffer solution. The spatial arrangement of the individual protein molecules on the surface of the virus capsid, also known as the capsomere, is clearly visible in the AFM image (ScanAsyst Fluid+ probe, k~0.7N/m).
PeakForce Tapping Imaging of the DNA Double Helix
Another ideal sample benchmark for PeakForce Tapping is DNA. It has been imaged extensively using AFM and was the first sample used to prove that TappingMode is ideal for imaging biomolecules. DNA comprises two polynucleotide strands, which form a double helix.
B-DNA is the “Watson-Crick” form of DNA, which is in the form of a right-handed helix with a helical repeat (pitch) of ~3.6nm, with minor and major grooves of widths ~2.2nm and ~1.2nm, respectively. This article enumerates a technique by which DNA’s secondary structure can be imaged using standard cantilevers and PeakForce Tapping.
In order to successfully image the DNA double helix, sample preparation is very important. Mica is normally used for AFM imaging. Mica however, at neutral pH has a negative surface charge that does not permit adsorption of the negatively charged DNA.
A number of methods have been deployed to overcome this, all the methods aiming at functionalizing the mica to develop a positive interface to which DNA can attach. In the year 2012, Leung et al. were successful in imaging the minor and major grooves of one DNA molecule using 1-5mM concentration of NiCl2 for DNA adsorption onto a mica surface.
This low concentration reduces adverse structural impacts on the DNA strands and minimizes surface contamination however, it also leaves the DNA bound loosely to the mica surface and creates a bigger challenge for high-resolution imaging.
The researchers used the same DNA immobilization approach as Leung et al., for imaging the helical structure of loosely bound DNA by deploying the low and accurately controlled imaging forces possible by PeakForce Tapping mode as achieved by Pyne et al.
PeakForce Tapping experiments were thus carried out on the Dimension FastScan Bio, MultiMode 8and BioScope Resolve™ atomic force microscopes (Figure 3) using MSNL-F, FastScan-D, ScanAsyst Fluid+ and ScanAsyst Fluid-HR probes, all of which have standard silicon tips.
Figure 3. Using PeakForce Tapping and standard AFM probes, imaging of the DNA double helix was demonstrated on all of Bruker's high-performance BioAFM systems: (left) Dimension FastScan Bio AFM, (middle) MultiMode 8 AFM, (right) BioScope Resolve AFM.
Corrugations were observed along the DNA strand corresponding to the minor and major grooves of the DNA double helix(Figure 4A) while performing PeakForce Tapping imaging on the MultiMode 8 in 10mM HEPES, 1mM NiCl, pH 7.4 (Figure 4A).
Figure 4A shows a high-resolution image captured by the BioScope Resolve operating on an inverted light microscope under identical imaging conditions, using ScanAsyst Fluid-HR probes. The image shows the widths of the alternating major and minor grooves, at 2.2 and 1.2nm, respectively.
In order to evaluate the mobility of the surface-bound DNA, continuous high-speed TappingMode imaging was conducted on the plasmid DNA, which was immobilized on the mica surface in 1mM NiCl2 (Figure 4B), using the FastScan-D probes and the FastScan Bio atomic force microscope that feature a standard silicon tip but a small cantilever.
Figure 4. (A) PeakForce Tapping image of a DNA plasmid taken in buffer solution using the Multimode 8 and MSNL-F probes (k~0.6 N/m) showing corrugation corresponding to the major and minor grooves of the DNA double helix. The inset is a high-resolution image of a DNA plasmid obtained using the BioScope Resolve operated on an inverted optical microscope and ScanAsyst Fluid-HR probes (k~0.05 N/m). The cross section taken along the strand, as indicated by the dotted line, shows the widths of the alternating major and minor grooves at 2.2 nm and 1.2 nm, respectively. (B) Time series of high-speed AFM images of the same type of plasmid DNA obtained in TappingMode showing that at low NiClconcentration some parts of the DNA remain immobile under continuous imaging (green arrows) while other parts of the same strand show a high degree of movement (red arrows). High-speed imaging was conducted on the FastScan Bio AFM using FastScan-D probes (k~0.2 N/m).
Along the DNA length, height variations were observed in the topography indicating twisting of the DNA strand (Figure 5A). This also suggests that the low Ni2+ concentration enables the DNA to retain a more physiologically relevant structure on the surface of mica.
Figure 5. (A) Topography image of a DNA plasmid captured in PeakForce Tapping mode in buffer solution. Local height variations are visible along the molecule as changes in color (white to red). (B) (i-iii) A DNA plasmid imaged at peak forces of 39, 70, and 193 pN, respectively, with the major and minor grooves of the DNA double helix visualized at higher magnification (insets). Color scales: 3 nm (for low magnification); 2 nm (for the insets). (iv) Height profiles measured across the DNA, as indicated by the dashed line in the inset of B, for different peak forces. (v) Measured height along the same section across the molecule (as iv) as a function of peak force. Figure 5(B) is reproduced with permission from Pyne et al.
The unique benefit of PeakForce Tapping over other intermittent contact modes is that it is possible to quantify the imaging force at all times.
Figure 5B(i-iii) shows the impact of force on AFM topography using PeakForce Tapping mode on the Multimode 8 by means of MSNL-F probes. In order to clearly show how compression of the DNA occurs with an increase in tip-sample force, the height scale is retained the same for all images.
At the least possible applied peak force of 39pN, the determined plasmid height is close to the 2nm DNA diameter as obtained from its crystal structure. Probably, because of the difficulty of tracking the molecule at these low forces, very small amount of corrugation is seen along the length of the DNA strand in corresponding high-resolution images, shown in Figure 5B(i).
About 20% molecule compression is observed at 70pN of the applied force, bringing down the determined height of the plasmid to ~1.6nm. As seen in the inset of Figure 5B(ii), at this force corrugation is seen clearly. The minor and major grooves become unclear beyond 100pN, (figure 5B(iii)) and just as in previous TappingMode AFM experiments in liquid, the height determined reduces to less than 1.5nm.
At this point, there is also a risk that the sample may dislocate from the mica surface demonstrated by the molecule movement as shown by the white arrow. It is seen in Figure 5B(v), that the height determined and the DNA diameter for applied forces of around 50pN or below agree with each other, while for resolving the secondary structure, a little more force may need to be applied.
A high-resolution image of a DNA plasmid captured using PeakForce Tapping on the FastScan Bio and FastScan-D probes at low force is shown in Figure 6. Corrugation corresponding to the double helix is shown in this image. To further study this structure, the scan size was brought down to image the smaller area highlighted by the white box.
Figure 6B show high-resolution images of this smaller scan area in which the minor and major grooves are clearly visible. In both trace and retrace images, the double helix structure is clearly seen and also in several subsequent scans shown in time order. The white arrows depict the scan direction.
The minor and major grooves show depth variations along the strand, which are reproduced between trace and retrace scans and also in further images (Figure 6C). This shows that the PeakForce Tapping mode not only resolves the submolecular features of the DNA double helix but also reproducibly captures variations in this helical structure.
Figure 6. PeakForce Tapping image of groove depth variations in the DNA plasmid topography obtained using the FastScan Bio AFM and FastScan-D probes (small cantilever and standard silicon tip). (A) Low-magnification AFM topography image of a plasmid showing corrugation. The white rectangle indicates the area imaged in B. (B) Higher-magnification trace (white arrow to right) and retrace (white arrow to left) images of this area showing corrugation consistent with the B form of DNA, for consecutive images. (C) Trace (solid) and retrace (dashed) height profiles taken along straight lines as indicated in B, closely following the backbone of the four plasmid scans and averaged over a 5-pixel (~0.5) width. The height profiles confirm the observed corrugation to be the alternating major and minor grooves of double helix structure and that these grooves vary in depth along the DNA strand. The height profiles have been offset by multiples of 0.6 nm for clarity.Color scales: 3.5 nm (A), 1.1 nm (B). Reproduced with permission from Pyne et al.
PeakForce Tapping mode offers accurate force control and easy quantification of the tip-sample interaction force, permitting imaging at forces below 100pN in order to obtain high-resolution images of soft biological samples in fluid environments.
By deploying the MultiMode 8, Dimension FastScan Bio, and BioScope Resolve atomic force microscopes from Bruker, it has been shown that the PeakForce Tapping mode is capable of imaging the major and minor groves of the DNA double helix on individual plasmids at high resolution.
In addition, It has been demonstrated that this kind of submolecular resolution can be consistently achieved without the need for specialized probes or specialized AFM designs.
This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.
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