Imaging of Biological Molecules with MAC Mode on the Keysight 7500 AFM

Table of Contents

Introduction
Instrumentation
Imaging of DNA in a Buffer Solution
Molecular-Level Visualization of Purple Membrane Proteins via MAC Mode Imaging
Conclusion

Introduction

In the field of biology, atomic force microscopy (AFM) can be utilized to work in aqueous environments. This is considered an interesting function and it makes this method suitable to measure or track biological molecules within physiological conditions.

With AFM imaging, the difficulty of probe oscillations and the interactions between the tip and sample force in solutions has been found to be relatively higher, compared to those under ambient conditions. For example, liquid structuring at a solid interface is a common phenomenon and this can considerably affect AFM’s operation, making the tip jump between varied stable configurations in the fluid’s ordered region.

A magnetic AC (MAC) mode, patented by Keysight Technologies, has been developed as a better alternative to AFM in liquid, where a thin magnetic coating is only applied to the cantilever portion of an AFM probe, and a direct tip oscillation occurs with the introduction of an alternating magnetic field. This approach removes the false resonances that can be observed in fluid tapping mode.

Oscillating the tip by directly applying a magnetic force, rather than triggering indirect mechanical excitation through an acoustic transducer, leads to a considerable amount of signal-to-noise for A/C mode AFM in fluids. This article shows some applications of the Keysight 7500 AFM for acquiring high-resolution MAC mode imaging of biological specimens in liquid.

Instrumentation

A high-performance instrument, the Keysight 7500 AFM/SPM microscope offers high resolution imaging with environmental control functions built-in. Keysight provides a patented magnetic AC mode (MAC Mode) as a system option. It is also quick and easy to swap imaging modes with the Keysight 7500 AFM/SPM microscope, thanks to the scanner’s easy-to-load and interchangeable nose cones.

The design and construction of the Keysight 7500 AFM have been improved to minimize mechanical noise, and at the same time provide exceptional performance. The scanner is compact and fully encapsulated. It offers a slot for (optional) preamps for CSAFM and STM operation, a built-in and highly reliable connector to interface with the control electronics, and easy exchange of the cantilever. All 7500 AFMs are supplied with closed loop position detectors, which have the lowest noise and provide excellent performance and convenience in imaging, without affecting the image quality and resolution.

Imaging of DNA in a Buffer Solution

The first example used to show AFM imaging in liquid is Lambda phase DNA. When DNA from a buffer solution containing Mg++ or Ni++ divalent cations is introduced to a mica substrate, the ions will create a charge bridge between the negatively charged siloxy groups bound on the mica and the negatively charged phosphate groups bound to the DNA backbone. This will result in a stable and consistent immobilization of DNA molecules on the substrate.

The quantity of DNA that can be adsorbed by the bare mica mainly relies on the concentration of DNA in the solution, buffer pH, buffer composition, and reaction/soaking time. An AFM topographic image is shown in Figure 1, illustrating an example of increased surface coverage of a DNA molecule. Separate DNA molecules with numerous configurations are seen clearly.

Figure 1. MAC mode imaging of lambda phage DNA in a buffer solution.

Molecular-Level Visualization of Purple Membrane Proteins via MAC Mode Imaging

The cell membrane of Halobacterium salinarium contains bacteriorhodopsin, which is a unique light-driven proton pump. Molecules of the bacteriorhodopsin adsorb on newly cleaved mica substrate to create a thickly packed 2D membrane. This membrane has a distinct purple color, and is usually referred to as the purple membrane (PM) in the literature. PM remains stable between pH 4 to 9 on the mica substrate, and is capable of forming patches of many hundred nanometers to many microns in size.

The membrane’s thickness measures roughly 5.5 nm, but somewhat changes based on pH. Additionally, contact mode AFM has been utilized for imaging PM in KCL solutions of varied levels of pH. Until today, contact mode AFM was used to obtain most of the high-resolution PM images seen. Despite this fact, contact mode imaging cannot be applied for a weakly joined bio-sample, because the AFM stylus disturbs the biomolecules during the course of scanning.

Dynamic force microscopy (DFM) techniques, such as MAC mode and tapping mode, are capable of reducing the lateral force applied on the surface of the sample and are more suitable for imaging biological samples that are soft and weakly joined together. Although, it is not easy to operate tapping mode in solution.

In the case of fragile samples like PM, the imaging force has to be quite low or else the PM’s lattice structure will be damaged easily. However, when using MAC mode an oscillating electromagnetic field directly drives the cantilever oscillation and the interaction force present between the sample and the tip can be controlled more precisely. As a result, high resolution images of the PM lattice structure can be obtained using MAC mode in solution.

The bacteriorhodopsin molecules, which are thickly packed, create highly structured 3D trigonal lattices in the PM. The MAC mode is used to acquire a topography image of a PM patch measuring roughly 200 nm in size. This is shown in Figure 2 (left). The MAC mode image clearly shows the hexagonal structure of the bacteriorhodopsin molecules with a repeating unit of approximately 6 nm and a corrugation of below 0.8 nm.The structure of the bacteriorhodopsin trimers in each repeating unit of the membrane shows a clear donut-like shape (Figure 2, right) at high magnification.

Figure 2. MAC mode imaging of purple membrane proteins in a buffer solution.

On meticulous analysis, the donut-like trimer structures are also revealed, which are probably joined by fibrous arms. This shows that the MAC mode AFM is a complimentary technique for examining the topography of weakly bound and soft biological samples, and can expose high-resolution single- or sub-molecular structures under physiological conditions.

Conclusion

This article has shown how the Keysight 7500 AFM can be effectively used to readily acquire high-resolution imaging of biological specimens at a molecular level. Here, the imaging of DNA and PM proteins in well-regulated buffer solutions were used as two examples.

This information has been sourced, reviewed and adapted from materials provided by Keysight Technologies.

For more information on this source, please visit Keysight Technologies.

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