Three-dimensional electron microscopy imaging of medical materials

Many samples of relevance to the medical materials industry are complex, containing mixed phases and interfaces buried within the sample. As an example one can consider what happens when a prosthetic device is implanted in an animal/human. The cells must stick to the device and spread and grow healthily. Studying the nature of this interface is therefore crucial, and it is important to find ways to image the three dimensional structure in its vicinity. Pills and tablets, as a means of delivering medicines, are another example. For these, the porosity of the tablet (which will depend on how the tablet was processed) and how the drug is distributed within the porous structure are key variables. The ability to image these structures at rather high magnifications, so that the detail of the structure is clear, is therefore crucial. Optical microscopy cannot always provide the level of detail required because of its limited resolution. However, conventional electron microscopy usually requires the use of high magnification and either ultra-thin samples (transmission electron microscopy) or only surface imaging can be performed (scanning electron microscopy). In this proposal we describe a new kind of electron microscopy which overcomes these problems, and opens up the way to build up three dimensional images of these complex medical materials. The technique uses a so-called Dual beam instrument, comprising both a conventional electron beam and also a focussed ion beam which can mill through materials. The ion beam cuts down through the material to expose buried interfaces. The electron beam, using a so-called environmental scanning electron microscope (ESEM) column, allows imaging of these exposed interfaces at high resolution. Because ESEM imagaing occurs in the presence of a low level of gas - and not an ultra high vacuum as in most scanning electron microscopes - insulators do not charge up under the electron beam. Thus the resolution is not degraded due to charging or the necessity of coating a sample with some conductive material. This approach will be developed for medical materials, both to develop appropriate protocols for this rather new approach, and also to solve specific problems to do with the porosity of tablets, and the nature of interfaces between spreading cells and an inorganic substrate.

The term Medical Materials covers a wide range of different types and applications. In all cases the materials tend to be heterogeneous in some way, with a need for careful understanding of this heterogeneous structure in order to optimise performance. Many techniques are used routinely to characterise this structure, and to relate to in vivo performance. However, there is often a need to obtain high resolution 3 dimensional images - at a resolution beyond that the confocal microscope can provide, and to examine in detail the interface of a prosthetic device consisting of an inorganic/metallic substrate and the cells which grow upon it. These are both approaches which provide significant experimental challenges. This proposal aims to develop appropriate protocols for examining the structure and performance of three broad classes of such medical materials using the rather new approach of Dual Beam Environmental Scanning Electron Microscopy, using an instrument just purchased under the BBSRC REI scheme. This technique permits the region of interest in the sample to be milled through using a focussed ion beam of gallium ions, thereby exposing buried interfaces. These can be imaged with the electron beam. Since this is an Environmental SEM, imaging is carried out in the presence of a low level of gas. The presence of the gaseous ions removes the need for insulators to be coated with a conductive layer, removing the need for post-milling processes, or translation of the sample. In this project we will develop robust protocols for this new technique, and apply the approach to study the porosity of tablets and the nature of interfaces between growing cells and a substrate. We will also apply the milling approach to cut out ultra-thin sections suitable for subsequent imaging in a TEM, where we expect less damage to be introduced in the vicinity of interfaces between hard and soft materials than is typically possible using ultramicrotomy.