One of the first biological molecules characterized by atomic force microscopy (AFM) was DNA. DNA imaging using AFM is often performed for characterizing the structure of DNA, protein interaction, dynamics and topology.
AFM images obtained initially presented DNA as a long featureless polymer without any signs of underlying helical structure. However, the use of enhanced force control and sharp AFM tips has made it possible to resolve the two oligonucleotide strands of the Watson-Crick double helix for single DNA molecules physisorbed on a micasubstrate, in a buffer solution.
These studies were made possible with current developments in AFM. With Bruker’s exclusive PeakForce Tapping technology, the DNA double helix can be imaged at high resolution and at measurable imaging forces, without using specialized probes or restrictive AFM designs.
Using AFM for Biological Research
Since the introduction of TappingMode technology in the early 90s, there has been a significant increase in the application of AFM for biological research. This technology allows the probe to oscillate at its fundamental resonance frequency and enables continuous adjustment of the vertical position of the tip (or sample) to keep constant amplitude of oscillation when the surface is being scanned by the probe.
The oscillation of the probe gives a tapping motion as the probe continuously moves in and out of the surface contact. The shear forces resulting from the previously used contact mode AFM are minimized by the intermittent characteristics of the tip-sample contact.
Indeed, this reduces the requirements on the extent to which the desired sample is rigidly fixed to a hard substrate, and also the need for fixation. As a result, the sample can be imaged under more physiologically relevant conditions.
Although TappingMode offers a number of benefits in characterizing the structure of biological samples, it does have limitations, such as delivering lower resolution images of biological molecules when compared to contact mode imaging.
It is important to maintain constant free oscillation amplitudes to achieve accurate setpoint amplitude measurement with respect to the tip-sample forces. However, this condition is not applicable for TappingMode in liquid as cantilever amplitude is a function of both the cantilever resonance and its convolution with mechanical resonances of the fluid cell.
These resonances tend to vary with changes in the composition, volume and shape of the liquid in the fluid cell throughout an experiment. This could alter the forces applied between the sample and tip, provided that the free amplitude of the cantilever varies. Therefore, it is difficult to achieve accurate determination and control of the imaging force during a TappingMode experiment.
Application of PeakForce Tapping Mode for Routine High-Resolution Imaging of Biomolecules
Bruker introduced the PeakForce Tapping AFM imaging mode in 2010. PeakForce Tapping quickly gained popularity following its introduction, finding application in the study of biological molecules.
PeakForce Tapping mode allows adjustment of the tip-sample distance in a sinusoidal motion at frequencies of 1kHz or 2kHz and at amplitudes below 100nm. Upon contacting the AFM probe with the sample surface, the tip-sample interaction is altered without disturbing the maximum force, or “peak force,” between the tip and the sample constant (Figure 1a).
By taking into account the movement of the probe with respect to the Z position, a curve at every pixel position on the sample surface can be achieved (Figure 1b).
Figure 1. (A) Modulation of AFM probe at low frequency in PeakForce Tapping Mode. (B) A force curve obtained at every position of the sample surface considering motion of probe in terms of Z position
PeakForce Tapping allows faster imaging of biomolecules to be taken than force-distance curve-based imaging modes. High frequency operation of PeakForce Tapping enables the generation of thousands of force curves per second.
PeakForce Tapping maintains low imaging forces to prevent damage in delicate samples and tips. It also facilitates more consistent and easier imaging in fluid, without the need to regulate the cantilever. Cantilever tuning is not required as PeakForce Tapping, unlike TappingMode, does not function at the resonant frequency of the AFM probe.
PeakForce Tapping also features the self-optimizing ScanAsyst imaging mode. The ScanAsyst mode allows auto-optimization of the imaging setpoint thereby avoiding setpoint drift due to cantilever deflection drift and/or resonance peak shifting. The setpoint drift is commonly observed in other AFM operating modes, including TappingMode and contact mode.
The PeakForce Tapping performance can be explained through the imaging of single virus capsids. A PeakForce Tapping image of a single herpes simplex virus is shown in Figure 2. From the image, the order of protein molecules in the form of 3D subunits over the virus capsid surface or capsomeres can be clearly observed. It should be noted that the virus particles were imaged without lateral stabilization, as individual and isolated particles.
Figure 2. 3D topography image of a single herpes simplex virus obtained in ScanAsyst mode in buffer solution
PeakForce Tapping Imaging of the DNA Double Helix
AFM has been widely used to image DNA. Furthermore, DNA is one of the first samples that demonstrated the capabilities of TappingMode for imaging biomolecules. A typical DNA molecule is made up of two polynucleotide strands forming a double helix. The section below shows a method of imaging the secondary structure of DNA using PeakForce Tapping and standard Cantilevers.
Sample preparation is critical for successful imaging of the DNA double helix. For sample preparation, DNA plasmid needs to be adsorbed on a relevant surface. Mica is one of the most common substrates for AFM imaging.
However, the overall negative surface charge of mica at neutral pH does not support adsorption of the also negatively charged DNA. A number of methods have been proposed to functionalize the mica so that a positive interface can be created for DNA attachment.
In 1995, Mou et al. solved the pitch of B-DNA by AFM as a periodic modulation of 3.4 ± 0.4nm. In their research, DNA was adsorbed onto a cationic supported lipid bilayer surface. The cationic supported lipid bilayer is deposited on a mica substrate. They observed the pitch of the DNA when the DNA strands were densely and uniformly packed on the bilayer surface.
Divalent cations serve as an alternative means of DNA absorption onto mica substrate. In this case, the adhesion can be regulated, to a certain extent, with respect to the cationic concentration in the solution, with Ni2+ being a convenient and effective choice. In 2012, Leung et al successfully imaged the major and minor grooves of a single DNA molecule with a 1 to 5mM NiCl2 concentration for DNA adsorption onto a mica surface.
Using the same DNA immobilization method as that of Leung et al., an attempt was made to resolve the helical structure of loosely bound DNA with the low and precisely controlled imaging forces enabled by PeakForce Tapping mode.
The PeakForce Tapping experiments were performed on the MultiMode 8, Dimension FastScan Bio, and BioScope Resolve™ atomic force microscopes (Figure 3) with the help of ScanAsyst Fluid-HR, FastScan-D, MSNL-F, ScanAsyst Fluid+ probes with standard silicon tips.
Figure 3. Schematic of Dimension FastScan Bio AFM (left), MultiMode 8 AFM (middle),BioScope Resolve AFM (right).
From PeakForce Tapping imaging on the MultiMode 8 in 10 mM HEPES, 1 mM NiCl2, pH 7.4, corrugations along the DNA strand were observed with respect to the major and minor grooves of the DNA double helix (Figure 4A) .
Continuous high-speed TappingMode imaging was carried out on the plasmid DNA immobilized on the mica surface in 1mM NiCl2 (Figure 4B) to analyze the mobility of surface-bound DNA. The analysis was carried out using the FastScan Bio atomic force microscope and FastScan-D probes. It is evident from the time series of high speed images that some regions of the DNA strand remain immobile under continuous imaging while other regions move across the surface.
Figure 4. (A) PeakForce Tapping image of a DNA plasmid taken in buffer solution using the Multimode 8 and MSNL-F probes. (B) Time series of high-speed AFM images of the same type of plasmid DNA obtained in TappingMode.
The topography along the DNA length showed height variations, likely indicating the twist in DNA strand (Figure 5A). This would also denote low Ni2+ concentrations that maintain a physiologically relevant structure of DNA on the mica surface.
Figure 5B (i-iii) shows the effect of force on AFM topography in PeakForce Tapping mode. The height scale of all images was kept constant to illustrate the extent to which the increasing tip-sample force has compressed DNA.
Figure 5. (A) Topography image of a DNA plasmid captured in PeakForce Tapping mode in buffer solution. (B) (i-iii) A DNA plasmid imaged at peak forces of 39, 70, and 193pN, respectively, with the major and minor grooves of the DNA double helix visualized at higher magnification.
A high-resolution image of a DNA plasmid imaged by PeakForce Tapping using FastScan-D probes at low force was shown in Figure 6A. A corrugation corresponding to the double helix is indicated in the image. Figure 6B shows the high resolution images of this smaller scan area that clearly shows the major and minor grooves of the strand.
The major and minor grooves exhibited changes in depth along the strand, which are further reproduced between trace and retrace scans (Figure 6C). This shows that PeakForce Tapping can resolve the submolecular features of the DNA double helix and also reproduce image variations in this helical structure.
Figure 6. (A) Low-magnification AFM topography image of a plasmid showing corrugation. (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. (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.
PeakForce Tapping enables accurate force control and simple quantification of sample interaction force. It allows imaging at forces below 100pN to achieve high-resolution images of soft biological samples in fluid environments.
The high-resolution imaging potential of PeakForce Tapping is illustrated by resolving the minor and major groves of the DNA double helix on separate plasmids with the help of Bruker’s MultiMode 8, Dimension FastScan Bio, and BioScope Resolve atomic force microscopes.
The consistent submolecular resolution achieved without specialized probes or dedicated AFM designs enables redefining the high-resolution imaging performance of AFM for biological samples.
This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.
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