Vertically orientated probes are an ideal tool to counteract the limitations of conventional atomic force microscopy (AFM).
Atomic force microscopy (AFM) has been evidenced to be an influential tool for imaging at the nanoscale. A basic diagram of an AFM setup can be seen in Fig. 1 (a).
Usually, a pyramidal, tetrahedral or conical tip, placed at the end of a horizontally mounted cantilever, is raster-scanned over the sample being investigated. A laser beam fixated on the surface of the cantilever is reflected onto a photodetector.
The placement of the laser spot on the photodetector moves as the tip is scanned over the surface, as a result of the vertical deflection of the cantilever, resulting from the variation in force between the tip and sample. An image of the surface is created from the position shift of the laser spot on the photodetector, as demonstrated on the computer screen in Figure 1, in which the color bar indicates the height of the features.
A number of limitations arise as a result of placing the cantilever almost parallel to the sample surface. First, the cantilever needs to be adequately stiff to extend horizontally over the sample surface, which restricts how low its spring constant can be and therefore, its sensitivity.
Furthermore, as the tip comes closer to the surface, the resultant shorter-range attractive forces experienced by the tip frequently grow larger than the spring constant of the cantilever. This can lead to the tip skipping into contact with the sample, known as a “jump-to-contact” event.
This can lead to damage to soft or delicate samples. Finally, as only the deflection of the cantilever in the vertical direction is subject to measurement, it is not attuned to the impact of lateral and shear forces.
Figure 1. Simplified schematic of an (a) AFM and (b) LMFM system.
Lateral Molecular Force Microscopy
To counteract these issues, a novel technique known as Lateral Molecular Force Microscopy (LMFM) has been developed by Dr. Massimo Antognozzi at the University of Bristol1, allowing for sensing using vertically-orientated cantilevers2.
The LMFM makes use of a different detection system to that used in standard AFM, known as “scattering evanescent wave” detection, as seen in Fig. 1 (b). An evanescent electromagnetic field is produced just over the sample surface, using a total internal reflected laser beam from an objective lens beneath the sample.
As the tip of the cantilever enters the evanescent field, the electromagnetic radiation is scattered. This scattered radiation is gathered with the objective lens and projected onto the photodetector, thus detecting the position of the tip.
As the cantilever is orientated vertically, it can be tip-less, with the free end of the cantilever going into the evanescent field. This allows for the detection of the true movement, rather than the movement inferred from the deflection of the cantilever.
The form of the cantilever is less important, and it is not necessary to have metal coating of the cantilever to increase light reflectivity, as there is no requirement for a laser beam to be reflected off the back of it.
Only the last few tens of nanometers of the free end of the cantilever interacts with the evanescent field, thus, the cantilever can be exceptionally small. The cantilever’s vertical orientation ensure that there is no “jump-to-contact” effect and subsequently, it is appropriate for the analysis of very soft samples.
It is not necessary for the cantilevers to be as stiff and therefore, they can possess very low spring constants, making them highly sensitive. NuNano has created tip-less, ultra-soft probes with spring constants in the range 0.003 – 6000 pN/nm, which are ideally suited for use in the described LMFM technique.
Applications of Lateral Molecular Force Microscopy
LMFM has been applied in a number of applications, which make use of its ability to calculate lateral and shear forces with nanometer resolution and femto-Newton sensitivity. These include:
- Imaging the motion of the biomolecular motor, Kinesin in the lateral force direction (2011)3
- Analyzing the mechanical properties of the adhesion protein, UspA1 when expressed on the cell surface of the bacterium, Moraxella catarrhalis (2011)4
- Shear force imaging of DNA in liquid (2012)5
- Imaging of self-assembled cages created by mixing coiled coil peptide modules (2013)6
- Analyzing the viscoelastic properties of membrane-free peptide/nucleotide protocells with small-molecule uptake (2013)7
- Directly monitoring the extraordinary optical momentum and force directed perpendicular to the wavevector, and proportional to the optical spin (2016)8
- Monitoring the evolution of hydration layers during metal nucleation in real-time (2017)9
References and Further Reading
- M. Antognozzi, A. Ulcinas, L. Picco, S. H. Simpson, P. J. Heard, M. D. Szczelkun, B. Brenner, and M. J. Miles, “A new detection system for extremely small vertically mounted cantilevers,” Nanotechnology, 19(38), 384002, 2008.
- J. A. Vicary, A. Ulcinas, J. K. H. Hörber, and M. Antognozzi, “Micro-fabricated mechanical sensors for lateral molecular-force microscopy,” Ultramicroscopy, 111(11), pp. 1547-1552, 2011.
- T. Scholz, J. A. Vicary, G. M. Jeppesen, A. Ulcinas, J. K. H. Hörber, and M. Antognozzi, “Processive behaviour of kinesin observed using micro-fabricated cantilevers,” Nanotechnology, 22(9), 095707, 2011
- C. Agnew, E. Borodina, N. R. Zaccaia, R. Conners, N. M. Burton, J. A. Vicary, D. K. Cole, M. Antognozzi, M. Virjib, and R. L. Bradya, “Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1
- binding,” PNAS, 108(37), pp. 15174–15178, 2011.
- R. L. Harniman, J. A. Vicary, J. K. H. Hörber, L. M. Picco, M. J. Miles, and M. Antognozzi, “Methods for imaging DNA in liquid with lateral molecular-force microscopy,” Nanotechnology, 23(8), 085703, 2012.
- J. M. Fletcher, R. L. Harniman, F. R. H. Barnes, A. L. Boyle, A. Collins, J. Mantell, T. H. Sharp, M. Antognozzi, P. J. Booth, N. Linden, M. J. Miles, R. B. Sessions, P. Verkade, and D. N. Woolfson, “Self-Assembling Cages from Coiled-Coil Peptide Modules,” Science, 340(6132), pp. 595-599, 2013.
- T.-Y. Dora Tang, M. Antognozzi, J. A. Vicary, A. W. Perriman, and S. Mann, “Small-molecule uptake in membrane-free peptide/nucleotide protocells,” Soft matter, 9(31), pp. 7647–7656, 2013.
- M. Antognozzi, C. R. Bermingham, R. L. Harniman, S. Simpson, J. Senior, R. Hayward, H. Hörber, M. R. Dennis, A. Y. Bekshaev, K. Y. Bliokh, and F. Nori, “Direct measurements of the extraordinary optical momentum and transverse spin-dependent force using a nano-cantilever,” Nature Physics, 12(8), pp. 731–735, 2016.
- R. L. Harniman, D. Plana, G. H. Carter, K. A. Bradley, M. J. Miles, and D. J. Fermín, “Real-time tracking of metal nucleation via local perturbation of hydration layers,” Nature communications, 8, 971, 2017.
This information has been sourced, reviewed and adapted from materials provided by Nu Nano Ltd.
For more information on this source, please visit Nu Nano Ltd.