Single molecules under the microscope
Submitted by rau on 03 March 2011
The idea of looking at single molecules or atoms has fascinated scientists for over a hundred years. This ambitious goal was first achieved in 1981 with the invention of scanning tunnelling microscopy, for which Gerd Binnig and Heinrich Rohrer at the IBM Research Laboratory in Rüschlikon, Switzerland, were awarded the Nobel Prize in Physics some time later, in 1986w1. This microscope has a severe limitation though: it works only on electrically conducting objects, so many interesting materials including biomolecules could not be studied. Binnig and his colleagues continued to search for better solutions, and in 1986, presented the atomic force microscope (AFM), which can be used to image both conducting and non-conducting materials.
Because this variety of forces can be measured with the AFM, it is very versatile and has led to an explosion in the number of scientists using the instrument – mostly but not only for material sciences and biology. In each case, the force causing the deflection is tiny and proportional to the tip’s distance from the surface.
Another major field of application for AFM in medical biology is the misfolding and aggregation of proteins such as α-synuclein, insulin, prions, glucagon and β-amyloid. These phenomena have long been implicated in degenerative diseases such as type II diabetes, Parkinson’s, spongiform encephalopathy (‘mad cow disease’), Huntington’s and Alzheimer’s. Here AFM has already provided important information on the nanoscale structure of the aggregates, and it is hoped that scientists will be able to use AFM to identify why the protein misfolds in the first place, and how it encourages surrounding proteins to adopt the same misfolded structure (Lyubchenko et al., 2010; for an explanation of prion misfolding, see Tatalovic, 2010).
The next big step will be using AFM for nanosurgery: introducing or extracting individual molecules from the cytoplasm of individual cells, to study cellular homeostasis or for subcellular drug delivery (Lamontagne et al., 2008; Müller et al., 2006).
Modified AFM tips can also be used as drills or pens: nano-milling removes material in the form of long curled chips (Gozen & Ozdoganlar, 2010), whereas dip-pen nanolithography is the controlled delivery of molecular or liquid ‘ink’. In chemistry and the life sciences, such technology is used to produce nanoscale sensors or, by the deposition of metallic, semiconductor and metal oxide nanostructures, functional nanocircuits or nanodevices (Basnar & Willner, 2009). This, combined with using the AFM tip to physically push nanometre-sized particles to a desired position, should pave the way for the miniaturisation of electronic circuitry and other structures.
Despite its vast number of applications – these are just a small sample – the possibilities of AFM are not yet exhausted. Future trends involve optimised tips and combinations with other techniques, for example to simultaneously determine surface structure and fluorescence or electrical properties (Müller et al., 2006). Speed is another issue: recently, an AFM has been developed with which biological processes such as chromosome replication and segregation, phagocytosis and protein synthesis can be imaged in real time, up to 1000 times faster than was previously possible (Ando et al., 2008).
Are you now itching to come up with your own applications for AFM? Then you might want to try and follow Philippe Jeanjacquot’s instructionsw2 for building your own instrument at school. It is a time-consuming project, but he and his students managed to create a feasibly low-cost microscope. There is one important catch though: you will need a vibration-free environment to set it up, such as a quiet cellar. If you can find that, your enthusiasm and ingenuity are the only limitations.
Al-Ahmad A et al. (2010) Biofilm formation and composition on different implant materials in vivo. Journal of biomedical materials research. Part B, Applied Biomaterials 95(1): 101-109. doi: 10.1002/jbm.b.31688
Ando T et al. (2008) High-speed AFM and nano-visualization of biomolecular processes. Pflügers Archiv: European journal of physiology 456(1): 211-225. doi: 10.1007/s00424-007-0406-0
Basnar B, Willner I (2009) Dip-pen-nanolithographic patterning of metallic, semiconductor, and metal oxide nanostructures on surfaces. Small 5(1): 28-44. doi: 10.1002/smll.200800583
de Farias Viégas Aquije GM et al. (2010) Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars. Plant Cell Reports 29(10): 1109-1117. doi: 10.1007/s00299-010-0894-9
Finlay JA et al. (2010) Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability. Biofouling: The Journal of Bioadhesion and Biofilm Research 26(6): 657-666. doi: 10.1080/08927014.2010.506242
Gozen BA, Ozdoganlar OB (2010) A rotating-tip-based mechanical nano-manufacturing process: nanomilling. Nanoscale Research Letters 5(9): 1403-1407. doi: 10.1007/s11671-010-9653-7. The full text article is freely available online.
Kimyai S et al. (2011) Effect of three prophylaxis methods on surface roughness of giomer. Medicina Oral, Patología Oral y Cirugía Bucal 16(1): e110-e114. doi: 10.4317/medoral.16.e110. The full text article is freely available online.
Kolind K et al. (2010) A combinatorial screening of human fibroblast responses on micro-structured surfaces. Biomaterials 31(35): 9182-9191. doi: 10.1016/j.biomaterials.2010.08.048
Lamontagne CA, Cuerrier CM, Grandbois M (2008) AFM as a tool to probe and manipulate cellular processes. Pflügers Archiv: European journal of physiology 456(1): 61-70. doi: 10.1007/s00424-007-0414-0
Lee GJ et al. (2010) A quantitative AFM analysis of nano-scale surface roughness in various orthodontic brackets. Micron 41(7): 775-782. doi: 10.1016/j.micron.2010.05.013
Lyubchenko YL et al. (2010) Nanoimaging for protein misfolding diseases. Wiley Interdisciplinary Reviews (WIREs). Nanomedicine and Nanobiotechnology 2(5): 526-543. doi: 10.1002/wnan.102
Müller DJ et al. (2006) Single-molecule studies of membrane proteins. Current Opinion in Structural Biology 16(4): 489-495. doi: 10.1016/j.sbi.2006.06.001
Padial-Molina M et al. (2011) Role of wettability and nanoroughness on interactions between osteoblast and modified silicon surfaces. Acta biomaterialia 7(2): 771-778. doi: 10.1016/j.actbio.2010.08.024
Poggio C et al. (2010) Impact of two toothpastes on repairing enamel erosion produced by a soft drink: an AFM in vitro study. Journal of Dentistry 38(11): 868-874. doi: 10.1016/j.jdent.2010.07.010
Tatalovic M (2010) Deadly proteins: prions. Science in School 15: 50-54. www.scienceinschool.org/2010/issue15/prions
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w1 – To learn more about the discovery of the scanning tunnelling microscope, which won Gerd Binnig and Heinrich Rohrer the Nobel Prize in Physics in 1986, see: http://nobelprize.org/nobel_prizes/physics/laureates/1986
Swiss scientists have developed the first AFM for planetary science, which forms part of NASA’s Phoenix mission to Mars. For a video introduction, see the Azonano website (www.azonano.com) or follow the direct link: http://tinyurl.com/6yguvb9
‘Universe today’ reports on the instrument’s success in the mission. See their website (www.universetoday.com) or follow the direct links: snow on Mars (http://tinyurl.com/6kp3rym) and Martian dust grains (http://tinyurl.com/64z6xrb)
If you enjoyed reading this article, why not browse the full collection of science topics published in Science in School? See: www.scienceinschool.org/sciencetopics
Dr Patrick Theer is a physicist who has spent most of his career developing microscopy techniques. After studying medical physics in Berlin, Germany, in Toronto, Canada, and in Guildford, UK, he delved into the field of non-linear optics for a PhD at the University of Heidelberg, Germany, studying the imaging depth limit in two-photon microscopy – an optical sectioning method that can provide information from very deep within scattering tissues. For his postdoctoral work, he moved on to the University of Washington in Seattle, USA, studying voltage-sensitive dyes using second harmonic generation microscopy. Currently, he works as a senior research assistant at the European Molecular Biology Laboratory in Heidelberg, developing a light-sheet-based fluorescence microscope for the study of embryonic development.
Dr Marlene Rau was born in Germany and grew up in Spain. After obtaining a PhD in developmental biology at the European Molecular Biology Laboratory, she studied journalism and went into science communication. Since 2008, she has been one of the editors of Science in School.
This article would be suitable for a wide range of science lessons – not only in physics but also when considering animal physiology or biomedical sciences, for example. Students can research atomic force microscopy and its uses further, as there is plenty of material on the Internet about the advantages and disadvantages of various microscopy techniques. They could also look up the group of scientists who invented the atomic force microscope (having won a Nobel Prize for a previous invention) and find out more about them and their work.
Potential comprehension questions include:
The article could be used with groups of older students or those most able to think creatively, perhaps for an extended writing task in conjunction with the film Honey I Shrunk the Kids (about a scientist working on a top-secret machine that miniaturises objects and – accidentally – people), to get the students to think about looking at single molecules. What would they like to use AFM for? Would they use their images as art or for scientific research? Would they want to use their knowledge to cure diseases or to see how beautiful science can be at this level?
Jennie Hargreaves, UK