A Guide to Using True Non-Contact Mode Atomic Force Microscopy (AFM) for the Imaging of Plasmids in Liquids

Research areas in agriculture, medicine, genetics, forensic sciences and molecular biology are just a few examples of fields that have been revolutionized by the discovery of DNA (Deoxyribonucleic acid). This biological molecule functions as a way of storing our genetic information, which is the cornerstone of every organism’s survival, playing a crucial role in reproduction, cell growth, and bodily functions.

DNA stores this information in the nucleus of each cell that makes up the organism and can be transferred from one cell to the next, as well as being passed down to offspring. Therefore, a thorough understanding of how DNA is structured is extremely beneficial in the comprehension of the mechanism of genetic information transfer.

True Non-Contact Mode Atomic Force Microscopy (AFM)

There is a range of techniques that can be used for DNA research, for example electron microscopy (EM). Unfortunately, this technique is not faultless, and there are difficulties surrounding sample preservation as a result of the long drawn out sample preparation process.

In response to this, scientists have developed a new technique called Atomic Force Microscopy (AFM). This technique allows researchers to take images of the sample whilst it is preserved in an aqueous environment, common in biological samples. This results in an improved quality of results and images that are produced with a higher resolution [1].

Park Systems offers a True Non-Contact imaging mode for AFM imaging which enables the collection of data of a sample’s topography in the absence of the probe end touching the sample, which is typical of other AFM imaging techniques, and therefore prevents damage to the sample as well as preventing the deterioration of the probe end.

Therefore, this program is the perfect partner for researchers needing to image samples that have surfaces which are delicate and easily damaged, instead of the more typical contact and tapping options.

The defining True Non-Contact imaging characteristic of Park AFM’s system is a result of the high-tech design of the Z scanner design. It retains the highest imaging resolution and precise topographical data collection, whilst ensuring probe end sharpness and minimal risk of affecting the surface of the sample [2].

Non-contact mode AFM topography image of plasmids. The segments indicated with yellow arrows have linear structures whereas those with green arrows exhibit supercoiled structures. Scan size 1 x 1 um.

Figure 2. Non-contact mode AFM topography image of plasmids. The segments indicated with yellow arrows have linear structures whereas those with green arrows exhibit supercoiled structures. Scan size 1 x 1 um.

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Experimental

Plasmids are a tiny DNA molecule that are often used in DNA research due to their ability to replicate independently of chromosomes. In the experiment, 45 nanograms of plasmids were scattered on a mica substrate and a Park NX10 AFM was used to capture images of them whilst suspended in liquid, True Non-Contact mode was engaged.

This experiment carried out investigations with a cantilever that had a reasonably low nominal resonance frequency of 110 kHz and a nominal spring constant of 0.09 nm.

The cantilever is manually oscillated near its resonant frequency for the duration of the scanning process. Recording the alterations in the size of the vibrations of the cantilever produced by Van der Waals attraction forces can be used to create the non-contact AFM image.

In order to keep the cantilever’s amplitude and distance consistent, the differences recorded are taken into account by feeding this information back to the AFM so that it can be adjusted accordingly. This feedback system is integral to how the non-contact mode records topography data of the sample surface by being able to adjust the Z scanner’s position [2].

Dampening effects of the sample being suspended in aqueous solution reduces the cantilever’s resonant frequency by a third of the result produced when measured in air, as well as a similar reduction in the measured amplitude. Aqueous samples also have additional peaks measured on a frequency sweep in comparison to data measured in air.

Therefore, it is important to select the correct resonant frequency prior to AFM imaging. This was done by choosing the highest peak that is closest to the region that is a third of the original resonant frequency. The amplitude was then set to approximately 0.9 nm.

A super luminescent diode (SLD) beam is reflected from the cantilever in order to transmit data to be fed back to the AFM. When using aqueous samples, the surface of the liquid is unstable throughout the measurement process and can cause the SLD beam to refract. As a solution to this problem, Park Systems created a liquid probe hand that is shielded which was also used in the experiment.

Results and Discussion

From the investigation, a high-resolution image of the plasmid sample was produced and processed using XEI software also created by Park Systems. It was initially anticipated that the sample would contain only uniform linear plasmids. But, the topography data, which can be seen in Figure 2, showed that the 1 by 1 µm being viewed actually consisted of a combination of linear and supercoiled plasmid DNA strands.

The supercoiled plasmid DNA strands were bigger and longer structures with diameters of about 19 nm. The smaller linear strands that were shown to be in a relaxed state had diameters of approximately 8 nm.

A line profile was also produced by XEI and provided detail about the height of the DNA strands. The data showed the supercoiled strands have a height of 1.6 nm in contrast to the height of the linear strand which was 1 nm.

During replication, the DNA is exposed to distortion by torsional stress, to protect itself, the DNA twists itself up in vivo. It is plausible that what was observed in the experiments of the shorter linear DNA is a similar phenomenon.

It can be suggested that the linear DNA supercoiled as a result of the torsional stress that occurred in the sample preparation process, with the end of the plasmid potentially sticking to the substrate more tightly than anticipated. This explains the bunching up and increased, supercoiled structure observed in the experiments, with the DNA increasing in size as the order of twisting increases [3].

Acknowledgments

Original authors: John Paul Pineda, Gerald Pascual, Cathy Lee, Byong Kim, and Keibock Lee Park Systems Inc., Santa Clara, CA, USA. Jason Kahn, The Department of Chemistry & Biochemistry, University of Maryland, College Park, MD, USA.

References and Further Reading

[1] A. Baro, et al., Atomic Force Microscopy in Liquid: Biological Applications. Wiley-VCH, Pages 233-237.

[2] J. Pineda, et al., True Non-Contact Imaging of Various Samples, Retrieved January 12, 2017, from http://www.parkafm.com/index.php/medias/nano-academy/articles

[3] H. Hansma, et al., Reproducible Imaging and Dissection of Plasmid DNA Under Liquid with the Atomic Force Microscope, AAAS, Pages 1180-1181.

This information has been sourced, reviewed and adapted from materials provided by Park Systems Inc.

For more information on this source, please visit Park Systems Inc.

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