Development of video atomic force microscopy for in vivo bioimaging of biological processes

Lead Research Organisation: University of Sheffield
Department Name: Chemistry


The ability to image biological entities is crucial to many key developments in science. Atomic force microscopy allows visualisation of living cells and systems without labels, in real time, with good spatial and temporal resolution. However a drawback of this technology, in order for it to be widely applicable to be used in high-throughput and for study of dynamics, is the time taken for image capture. We are at the forefront of development of VideoAFM, which will increase the speed of imaging by 1000 fold. This will revolutionise the use of the technology in many biological applications. This unique project will fully integrate technology development with addressing real biological problems in the area of microbial cell surfaces. In particular we will examine the surface properties of the major pathogen Staphylococcus aureus, which allow it to interact so successfully with its human host. A direct collaboration between the investigators and a commercial manufacturer of the technology will allow practical outcomes to be converted into real products within a short timespan. Thus the project will overcome existing limitations to give a widely useful technology for biological science, which will be exemplified by the applications carried out during the project.

Technical Summary

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.


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Description Conventional atomic force microscopy allows high resolution (down to the molecular scale) imaging of surfaces in essentially any environment. This makes it a powerful tool for biology, where the relationship between structure, form and function lies at the heart of many current questions. However, one of the drawbacks of the technique is the slow time that it takes to collect data, making rapid processes inaccessible for imaging, and routine data collection rather slow. This project aimed to develop our VideoAFM technology to provide a new and flexible tool for use in biological applications. However, biological systems are soft, and the technique was developed for imaging hard materials, so the approach required development. To do this we slowed it down from its true video-rate imaging to around 1 frame/s, fast enough to greatly improve data collection, but slow enough to keep the sample in one piece. This approach also allowed very large high speed scans to be performed (the largest of any high speed AFM developed to-date), so enabling good sampling statistics.

We applied our new approach, in conjunction with conventional AFM imaging for ultimate resolution, to elucidate the architecture and growth behaviour of a number of bacteria, focussing on the important human pathogen Staphylococcus aureus and the soil bacteria B. subtilis. In both cases new and unexpected features of the bacterial cell wall were revealed, and our new model has already been used in two microbiology text books. In S. aureus a new feature of the cell wall was revealed which may allow the bacteria to determine its division plane, giving it the characteristic 'bunch of grapes' morphology from which it gets its name. We also developed a new, passive and non-chemical, way to immobilise the bacteria, allowing them to be imaged during cell division and while excreting material that may be important for the formation of biofilms, which play a role in the pathogenicity of these bacteria, e.g. by coating surgical devices.
Exploitation Route The large area scanning capability may find application in semiconductor device fabrication for process control. In the longer term it may be possible to adapt this to applications in soft and biological matter in an industrial context. We are working with Infinitesima Ltd, one of the grant partners, to realise the large area scanning capability in a commercial environment.

We have obtained further funding from BBSRC to explore the biological aspects of the work further.
Sectors Electronics,Manufacturing/ including Industrial Biotechology

Description Further development of high speed AFM informed the developments made by Infinitesima Ltd
First Year Of Impact 2008
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic