The Astbury Centre for Structural Molecular Biology
Gallery and Structures
The Astbury Society
Scanning Probe Microscope Facilities
Within the Astbury Centre, in the Department of Physics and Astronomy, there is a broad range of scanning probe microscopes (SPMs) including scanning tunnelling microscopes (STMs), atomic force microscopes (AFMs), magnetic force microscopes (MFMs) and scanning near-field optical microscopes (SNOMs).
Atomic force microscopy is particularly suitable for imaging and manipulating biomacromolecules, cells and tissue (see article below) under physiological conditions.
Dr Neil H Thomson firstname.lastname@example.org
If you would like to discuss your imaging or force spectroscopy application then please contact Neil by email.
Dr Neil Thomson using one of the Digital Instruments Multimode AFMs
Scanning Probe Microscopy Tutorial
For a full practical guide to AFM go to http://www.pacificnano.com/afm-tutorial.html
The Atomic Force MicroscopeA Tool for Imaging and Manipulating Biomolecules under Native Conditions
Microscopes have historically been tools of great importance in biological science. The atomic force microscope (AFM) is one of a family of scanning probe microscopes which has grown steadily since the invention of the scanning tunnelling microscope by Binnig and Rohrer in the early eighties for which they received the Nobel Prize for Physics in 1986. The AFM uses a cantilever usually made from silicon or silicon nitride with a very low spring constant, on the end of which a sharp tip is fabricated using semiconductor processing techniques. Figure 1 shows electron micrographs (courtesy of Veeco Metrology) of the triangular and springboard shaped cantilevers that are used and a close up of the sharp tip which is used for imaging. When the tip is brought close to a sample surface the interatomic forces between them cause the cantilever to bend and this motion is detected optically by a laser beam which is reflected off the back of the cantilever. If the tip is scanned over the sample surface then the deflection of the cantilever can be recorded as an image, which in its simplest form, represents the three dimensional shape of the sample surface. Many variants of the technology now exist which use special tips to probe the electric, magnetic or thermal properties of surfaces.
Figure 1 An electron micrograph of typical AFM cantilevers showing the triangular and 'springboard' designs of contact mode and tapping mode probes. A close up of the tapping mode cantilever shows the sharp tip used for imaging.
The resolution of AFM depends mainly on the sharpness of the tip which can currently be manufactured with an end radius of a few nanometers. Atomic resolution is easily obtained on relatively robust and periodic samples. Soft samples however, particularly biological samples, provide a more difficult surface to image because the forces exerted by the tip during imaging can cause deformation of the sample. The problems involved with imaging soft samples have been overcome to a large extent by the introduction of 'tapping mode' AFM imaging. Instead of maintaining a constant tip-sample distance of a nanometer or so, the cantilever is oscillated in a direction normal to the sample resulting in only intermittent contact with the surface. This greatly reduces the lateral forces being applied in the plane of the sample which are responsible for most of the damage as the tip is scanned. The AFM is capable of better than 1 nm lateral resolution on ideal samples and of 0.01nm resolution in height measurement.
There are some significant advantages of AFM as an imaging tool in biology when compared with complimentary techniques such as electron microscopy. Not only does AFM achieve molecular resolution but the technique requires almost no sample preparation and, most importantly, can be performed under fluids permitting samples to be imaged in near native conditions. The fluid may be exchanged or modified during imaging and therefore there is the potential for observing biological processes in real time, something which electron microscopy is not currently able to achieve. Several instruments are commercially available for around £100k and in general the technology is straightforward to use and occupies only a small table top.
There have been many studies of biological materials using AFM in the few years since its conception. Examples include nucleic acids and their complexes with proteins, two dimensional protein crystals and individual isolated proteins, membranes and membrane bound proteins and living cells.
Figure 2 Tapping mode AFM image of a fibril formed by the aggregation of the protein, b2M (Scale bar is 200 nm).
Figure 2 shows a high magnification tapping mode AFM image of fibrils of the protein b2M. These fibrils are formed by the aggregation of proteins and such structures are implicated in diseases like Alzheimer's and Parkinson's, and in the case of b2M, dialysis related amyloidosis. This AFM image is of a mature fibril formed from a mutated version of the b2M protein and clearly displays a left handed periodicity. The significance of these periodic structures, the process of their formation and their pathological effect are still largely unknown.
Figure 3 Contact Mode AFM image of a DMPC/DSPC(1:1) bilayer showing phase separation.
Figure 3 shows an AFM image taken under PBS buffer at room temperature of the phase separation in a phospholipid bilayer of a 1:1 mixture of DMPC/DSPC. The dark areas are fluid phase and the lighter areas are gel phase lipid. There is an approximately 10 Angstrom difference in height due to the difference in packing in the gel and fluid phases. The white dot is probably a lipid vesicle that has not unrolled.
Figure 4 A 4x4 micron phase image of material lifted from a section of human brain tissue.
As well as the imaging of molecules on a flat substrate, AFM is capable of imaging cells, tissue sections or material extracted from such samples. Figure 4 shows a 4x4 micron tapping mode phase image of a sample of material from human brain tissue prepared by touching a piece of clean mica on the fresh tissue surface. The diagonal structure to the left is a neurofilament. In a phase image the different colours correspond to different mechanical properties of the sample. The surrounding material in this sample shows three main phases - it is hoped that structures such as amyloid plaques will show up clearly in such images.
To see some other excellent examples of what can be achieved with the AFM and detailed information about its operation, visit http://www.di.com
In addition to the very high potential of the AFM to provide high resolution images of biological samples and to monitor conformational changes and biomolecular processes in real time under native conditions, the instrument is also capable of manipulating molecules and measuring the strength of molecular interactions with pico-newton sensitivity. It is relatively straight forward to attach biomolecules to the AFM tip so that the interaction between these molecules and the sample can be measured. In this case the sample may be a protein or nucleic acid, or a solid or cell surface. Perhaps one of the most exciting demonstrations of the potential of the AFM to measure biomolecular interactions has been the recent examples of the mechanical unfolding of single proteins. Several groups have shown that a protein, held between the tip and a solid support by its two terminal groups can mechanically unfolded by pulling with the AFM.
Understanding the mechanisms of protein folding has been one of the key questions in structural biology over the past 30 years and remains one of the most significant post-genome challenges. Over the last few years, the folding transitions of various proteins have been studied using a variety of spectroscopic methods which have led to detailed insights into the conformational properties of the biomacromolecules. However, the key issue in folding - how the amino acid sequence of a protein encodes its final structure and therefore its function - remains unresolved. The collaboration between Physicists and Biologists to develop new experimental methods that can access novel data concerning protein structure and dynamics is critical to unravelling the protein folding problem and it is just one example of the exciting challenges there are to be found for Physicists in Biology today.
At Leeds University over the past few years we have been developing the biochemistry and molecular biology to synthesise poly-proteins - a repeat series of the same protein like beads on a string - in order to study their response to an externally applied force field. We have also developed techniques that allow us to pick up these proteins and unfold their complex structures with the AFM. Figure 5 is a cartoon of just such an experiment. The poly-protein is held between the AFM flexible cantilever and a solid support. As the probe is moved upward the forces required to pull the protein structure apart cause a bending of the cantilever and can therefore be recorded as a function of extension. A typical force-extension curve is shown in the inset. At point A, the tip picks up the poly-protein by one domain and the whole protein is extended. At B, a critical force is reached at which the least mechanically stable domain ruptures. The tension is released by the creation of a length of unfolded polypeptide chain and the cantilever returns almost to its rest position. In part C, as the ends of the protein are moved further apart the unfolded domain is extended and the applied force gradually rises until a second domain unfolds.
By analysing many protein unfolding events such as these under a range of experimental conditions it is possible to determine a number of key parameters such as the folding and unfolding rate constants in the absence of the applied force field, the size and positions of energy barriers that control these rate constants and make comparisons of the mechanical unfolding process with more traditional chemical methods. In addition, the fact that these experiments are carried out on single molecules allows a direct comparison with computer simulations of protein dynamics which invariably only deal with a single molecule. Eventually, these experiments will lead to a deeper understanding of protein folding mechanisms which may ultimately allow us to treat diseases of protein misfolding of which there are many, and design new synthetic proteins to have unique functions.
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