The recent development of VideoAFM has provided a 1000 fold increase in frame-rate compared to conventional AFM technology. However, to bypass the limitations of the standard approach to AFM, we have had to completely reappraise the way in which the sample is scanned and the way in which the AFM tip tracks the sample surface. This has introduced several issues for the application of the technology to biological systems, and it is the purpose of the current application to address these issues and test the technology through application to several key areas in our understanding of the human pathogen S. aureus. Imaging in liquid is central to bio-applications. We will develop VideoAFM for stable use in liquid, and methods for the rapid control of sample environment to facilitate the in-situ observation of processes with nanometre spatial and sub-second temporal resolution. This will allow direct, real-time imaging of processes such as the action of antibiotics on living cells. Imaging with chemical specificity is one of the strengths of conventional AFM, but is currently beyond the capabilities of VideoAFM. We will develop a technique for rapidly monitoring the frictional force component, and apply this to the in-situ mapping of cell-surface physical properties at the molecular scale. We will also develop a combined video-rate and conventional AFM approach to the measurement of surface interaction forces, using VideoAFM scan rates to find areas/times of interest and automated switching to conventional AFM to then locally probe the functionalised tip / surface interaction. High throughput methods require large areas to be rapidly imaged with molecular resolution. We will combine conventional and resonant scanners to allow macroscopic areas to be imaged with VideoAFM through a tiling process, so that hundreds of microns can be imaged in tens of seconds at nanometre resolution, allowing comparison of surface structure over large populations of bacteria. This project is in collaboration with Prof Simon Foster and Infinitesima Ltd.
The Snomipede project is funded by the Basic Technology Programme and unites researchers at the Universities of Sheffield, Glasgow, Manchester and Nottingham. The objective of the project is to develop a massively parallel near field lithography device that combines the parallelism of the Millipede, developed by researchers at IBM, with the powerful capability for molecular patterning provided by the technique of scanning near-field photolithography, developed by the Leggett group over the past few years. See also the SNOMipede website.
Many bacteria responsible for disease secrete proteins which are a method by which the bacteria invade the host and disable its defences. Atomic force microscopy (AFM) is an imaging technique that can obtain images of individual proteins within membranes or on surfaces under physiological conditions, and potentially follow processes at a molecular scale in real-time. Recent developments by the applicant allow considerably faster imaging, providing an opportunity to follow the process of pathogenic protein invasion in real time, gaining a new understanding of the role played by specific proteins. For instance, it should be possible to follow an individual protein secreted by the bacteria responsible for meningitis and septicaemia as it enters the cell membrane of a red blood cell, image the resultant changes in cell shape, and gain a new understanding of role played by the protein in the cell’s death. This grant aims to transfer expertise from physical science into medicine, providing access and expertise in equipment not previously applied to a medical problem. A physical scientist researcher will be physically re-located into the School of Medicine, providing them with considerable new training and giving them a unique skill set for applying AFM technology to these important systems. It will also give the physical scientist applicant sufficient time to become well acquainted with the medically relevant biology, providing a sound basis for the development of new AFM related technologies of wider application to medicine.
Atomic force microscopy (AFM) is one of the key tools for nanotechnology and nanoscience. We recently developed a way of increasing its scan speed more than 1000 fold, allowing video rate imaging for the first time. To do this we combined resonant scan stages capable of line rates of tens of kHz with a novel 'passive mechanical' feedback method in which the AFM tip is forced to respond to the sample surface at MHz frequencies. Although images were obtained, they were difficult to interpret and the new method of scanning departed so far from the prior art that simple interpretations of force were no longer applicable. The aim of this project was to rectify this, putting the new technique on a sound experimental and theoretical footing.
We took two approaches to this - using finite element modelling to model the cantilever response to the surface and hence calculate the forces applied to the surface both vertically and laterally, and using complementary experiments to verify the models, tracking the cantilever response at multiple (three) points along its length in real time. The FEM analysis was remarkably successful, reproducing faithfully the VideoAFM cantilever behaviour and allowing reconstruction of images that corresponded very well with the real data. VideoAFM images are the result of the transient cantilever response, and the value obtained at a point in the image depends not only on the slope and height of that point, but also on the previous trajectory of the probe both because of its angle when it encounters the new feature and energy stored in the cantilever. It is therefore not possible to simply transform a VideoAFM image into the true topography – instead the full response of the cantilever to the entire surface must be considered. Model outputs such as forces can now be used to help further developments of the instrument. The high frequency force response led to the development of novel cantilever designs better suited to high frequency imaging – i.e. with reduced stiffness when responding rapidly. Cantilever damping was also found to be particularly important and we developed a rigorous understanding of the behaviour of the existing cantilevers and made progress towards obtaining new geometries with improved performance. Publications have been submitted in these areas (delayed due to the need to clear with our industrial partners). In summary the VideoAFM image contrast can be well understood through a continuum mechanics treatment of the transient response of the AFM cantilever to the sample surface. Peak tip-sample forces of 100s of nN were typically found for surfaces with sharp topographic features ~60 nm in height, with lateral forces depending critically on the steepness of the effective surface (i.e. sample convoluted by the tip).
To accurately determine the cantilever trajectory as it tracked the surface at video rate we built a VideoAFM on an optical bench, allowing multiple lasers to be focussed onto different positions along the cantilevers length, the data being collected by a multi-channel VideoAFM built for us by Infinitesima. The results showed good agreement with our simulated data of cantilever deflection. We are still ensuring that IP in this area is protected prior to publication. Novel detection methods were also developed which promise to allow higher bandwidth data collection in the future.
Expanding the VideoAFM technique to allow surface property determination is key to widening the applicability of the method. We developed an approach in which multiple images are collected with varying force of the same area, effectively mapping out the force-volume behaviour of the material. Simple difference images give an indication of varying properties across the sample surface. The technique works, but is hindered by the VideoAFM contrast mechanism. We have plans for a ‘true height’ VideoAFM which should solve this issue, allowing near video rate surface mechanical mapping.
The project aimed to use state-of-the-art atomic force microscopy techniques to gain a greater insight into how polymers nucleate at the molecular level. In-situ imaging of the nucleation of isolated polypropylene droplets allowed the nucleation behaviour of this industrially significant polymer to be probed at the nanometre scale for the first time. Different nucleation regimes were identified depending on the thickness of the polymer film, with possible implications for the processing and use of polymers in nanoscale devices. The length scale over which the volume dependence of nucleation rate has been explored was extended by several orders of magnitude in this material. It was also possible to image, for the first time, the growth and subsequent re-organisation on heating of the elusive partially ordered or ‘smectic’ phase of polypropylene. The droplet technique was found to be a particularly powerful one for the investigation of polymer crystallization at high supercoolings and under conditions of spatial confinement. This work has led to a collaborative grant with Nanjing University in China, allowing inter-cultural exchange as well as further developing our lead in this area of real-time AFM imaging of polymer processes.
An experimental technique was developed for tethering individual polyethylene molecules (another polymer of great industrial importance) to the AFM tip, allowing the changing conformation of the molecule on a nucleating surface to be investigated by pulling the chain away from the surface and monitoring the ensuing force with the AFM cantilever. We believe this approach allows the changes in molecular conformation that occur during crystallization to be probed at the sub-molecular scale in a way that is not possible by conventional imaging methods. This is believed to be the first time that a phase transition has been followed through this ‘force spectroscopy’ technique in a synthetic polymer, and opens up a range of possibilities for future studies.
A newly developed video rate atomic force microscopy was also applied to the crystallization process, and the highest frame rates were obtained for any process followed with a scanning probe technique. This will have wide reaching implications for the whole of the nanotechnology community as it has proven the worth of a new and potentially revolutionary nanoscale imaging tool. This video rate study provided new insights into how polymer structures evolve rapidly under conditions closer to those found in real processing than has been previously possible at this resolution, as well as answering long standing questions relating to the structure formation of large crystal aggregates known as spherulites. The data have been taken up by the UK SME that sells and develops the microscope and has played an important role in their marketing, showing how fundamental scientific research can benefit high technology companies.
The grant was a ‘First Grant’ and as such also played a role in developing the career of a new academic. Both through the related grant income that has derived from this research, and from the invited conference talks and exposure that it has given, the grant has been an undoubted success in this respect.
The fellowship aimed to use newly developed atomic force microscopy (AFM) techniques for following processes in-situ in real time to gain new insights into how industrially significant polymers crystallize. At the same time, it aimed to leave the research fellow as an independent and internationally recognised researcher both in the field of polymer physics and scanning probe microscopy (SPM). It was remarkably successful on all fronts. AFM was used to directly image crystal growth in a wide range of different materials, providing direct proof of many previous deductions, discovering several new phenomena, and shedding light on the question of intermediate phases during crystal growth. In particular, although no evidence was found for semi-ordered phases occurring between the melt and crystal state, behaviours that contradicted classical theories were also observed such as sporadic growth rates at the lamellar scale, and fluctuations in growth front over distances of tens of nanometres. This has led to a more accurate parameterisation of what any possible intermediate phases must be like. A wide range of collaborations, both national and international, were set up through the period of the project, providing access to many exemplar materials. At the same time a complementary set of x-ray scattering experiments were performed that have helped to improve understanding of exactly how crystal thickness and chain length relate to crystal stability.
In order to facilitate the in-situ AFM experiments, an extensive instrument development project was undertaken and this proved highly successful, leading to the development of an instrument capable of obtaining nanometre resolution images approximately 1000 times faster than conventional AFM. This new instrument, VideoAFM, utilises two revolutionary new approaches, resonant scanning, and a passive effective feedback system that allows a surface to be scanned with nanometre resolution while the AFM tip is traversing the surface at a velocity of more than 20cms-1. This new technology is now being marketed by the company, Infinitesima Ltd., which the research fellow helped to set up. The capability of scanning at truly video rates has opened up a wide range of research (as well as commercial) opportunities, in particular in biological systems, and high-throughput surface analysis, which are now being explored.
The research fellow now has an established group (3 post-doctoral research associates and 4 PhD students) with a permanent position at a leading UK University (University of Sheffield). He has obtained significant grant support (more than £800k) as Principal Investigator, as well as being deeply involved with collaborations both within Sheffield and abroad. His international reputation has led to a large number of invited talks at international conferences, as well as further collaborations both with other academics and industrialists. His research has also broadened into the life sciences where it is hoped that the technology that has been developed can make a significant impact to address quality of life issues.