DRUG DISCOVERY Developments in electron microscopy Electron microscopy is an extraordinarily powerful technique which will continue to evolve well into the 21st century. Paul Ansell, Hitachi Scientific Instruments Although a oth scanning and transmission electron Field emission sources number of microscopes (SEMs and TEMs) are used Although a number of factors influence the extensively in pharmaceutical applications. factors influence B resolution of an electron microscope, the electron The range of applications is diverse, from source plays a major part. In simple terms, the the resolution of ultrastructural pathology, ultrastructural localisation smaller electron the beam and the greater the an electron of antigens and high-resolution studies of viruses current density within that beam, the smaller the and proteins, to investigations of drug delivery microscope, the details that can be resolved at high magnifications systems such as aerosols, ointments, creams and with better image quality. Original electron sources electron source tablets. SEMs are used to examine the surface of a for both SEMs and TEMs were thermionic tungsten plays a specimen and produce characteristic ‘3-dimensional’ emitters - literally a heated tungsten filament images of the sample, together with a much greater which emits electrons. The next development major part depth of field than can be obtained using optical was the lanthanum hexaboride emitter, which microscopes. Transmission electron microscopes produced a brighter electron source than the are more analogous to light microscopes, with the thermionic tungsten source. However, the key electron beam passing through a thinly sectioned advance over the last 10-15 years has been the sample with the image projected onto a phosphor development of stable field emission electron guns viewing screen. which have a brightness some three orders of The capabilities of electron microscopes have magnitude greater than the standard emitter. In increased remarkably since they became available addition, field emission sources have low energy commercially, and many of these have benefited spread and smaller source diameters. A schematic the pharmaceutical industry. Equally important diagram is shown in Figure 1. has been the development of energy dispersive Field emission systems are more costly and X-ray analysis (EDX) techniques, which allow the do require much more demanding vacuum elemental composition of the materials being conditions. A conventional tungsten emitter imaged to be determined. EDX systems can be operates in a vacuum environment of around 10-5 used with both SEMs and TEMs, and provide Pa, whilst the field emission source requires an a truly powerful supplementary tool to the operating vacuum in the order of 10-7 Pa. Thus microscopist. However, this subject could warrant field emission microscopes have more complex an article in its own right, so this article will pumping and valving arrangements for the electron concentrate on some of the developments on the columns. The S-4700 field emission SEM is microscopy side that have particular significance. shown in Figure 2. Field emission sources have These are: field emission electron sources, digital particular importance for SEMs, because they image acquisition and frame averaging, and the allow much lower electron beam accelerating variable pressure SEM. voltages to be used on samples. This offers a 40 Innovations in Pharmaceutical Technology DRUG DISCOVERY Figure 1. Principles of cold cathode field emission electron sources. ... the key number of benefits, such as reduced sample damage, reduced likelihood of sample charging, advance over and reduced penetration of the electron beam into the last 10-15 the sample surface, revealing greater surface detail. years has Reduced sample charging is important in pharmaceutical applications since many of the been the the specimens under study are non-conducting and development charge up when exposed to the electron beam. of stable field This produces unwanted image artefacts and impairs image quality. emission Another important use for field emission electron guns ... sources in pharmaceutical applications is in immunogold labelling experiments. This is a technique used for the localisation of specific Figure 2. S-4700 field emission SEM from antigens. Samples are immuno-stained using an Hitachi Scientific Instruments. antigen-specific primary antibody followed by a gold-labelled secondary antibody, which localises to the required antigens within ultrastructural components and organelles of cells. These gold particles are extremely small (0.8nm - less than 20 gold atoms), so the high brightness and resolution of the field emission source is essential. Field emission sources are also important for TEM studies of ointments and creams, where it may be necessary to image ultra-structural details down to molecular dimensions. Digital imaging and frame averaging Scanning electron microscopes work by scanning an electron beam over the surface of a sample in synchronisation with scanning a display monitor. Electrons emitted from the surface as the beam interacts with it are collected, and the resulting signal is used to produce the image on a viewing screen. Traditionally, the slower the scan, the better the quality of the image, as the beam spends Figure 3. H-7600 TEM showing digital imaging. longer at any one point on the surface, so more 42 Innovations in Pharmaceutical Technology DRUG DISCOVERY electrons are emitted and the signal-to-noise the samples with a thin layer of a conductive ratio improves. Micrographs could be recorded material such as gold; it was not always possible, photographically by slowing the scan down even however, to get uniform coatings on rough surfaces more, and a good quality micrograph could take and sometimes the gold layer could itself mask several minutes to produce. Slow scanning may surface detail of interest. Scanning at TV rates have improved the signal-to-noise ratio, but the significantly reduces the charging effects, which long refresh rates made it difficult for the operator meant that such samples could now be examined to optimise the focus and other operating without coating. The image held in the framestore parameters, or to move the sample around to was indeed a digital image but, in the early locate the precise area of interest. days, it had to be transferred to film to produce The introduction of TV rate scanning was a a hard copy. significant development, because it resulted in The PC revolution of the 1990s changed real-time imaging which was useful for focusing all that. The development of large volume, Another and moving the specimen, but under high resolution electronic storage media allowed images to be important conditions, the images could be very “snowy” saved electronically and printed using video use for field or noisy. In the mid-1980s, however, the digital printers - removing the need for a dedicated image store was introduced into scanning electron photographic recording unit. The framestores grew emission microscopy. This essentially consisted of a in pixel resolution capability from the early 640 x sources in framestore, with integrating electronics, which 480 (approximately) pixels to typical present day pharmaceutical meant that frame after frame was added into the values of 2560 x 1920 pixels - the equivalent of the total and then averaged. This reduced noise old photographic resolution. Digital imaging has applications is significantly and users could see their images now also been extended to transmission electron in immunogold magically appearing out of the snow! An added microscopy, with cameras producing digital images boon was that the “cleaned up” image was held in from the phosphor screen. This is illustrated in labelling the framestore, and this could be transferred Figure 3. The additional benefit that digital experiments to film very quickly. imaging has brought is the ability to perform a host of online image processing routines. This can range from image enhancements (filtering, sharpening, smoothing, differentiation and so on) to improve the appearance of the image or to emphasise a particular feature of interest, to image analysis measurement functions such as area, diameter and roundness measurements for individual features such as viruses. The automatic identification of viruses is shown in Figure 4. Variable pressure scanning electron microscopy In pharmaceutical applications, many of the samples are of tissues, or of creams or ointments, for example, which all contain liquid to a greater or lesser degree. Sample preparation for the examination of such specimens in the SEM has always been an involved process. Outgassing of water vapour in the high vacuum environment of Figure 4. Automatic identification of negatively the SEM can both degrade the performance of the stained adenoviruses in the TEM using instrument, and lead to deformation or collapse of digital techniques. the structure under study. In addition, the non-conducting nature of the organic material Apart from making the instruments easier to made it difficult to obtain high resolution, high use, this development had particular benefits for magnification images without further preparation. the pharmaceutical industry. Many pharmaceutical To overcome these difficulties, biological samples - such as powders, tablets and so on - are specimens were traditionally subjected to sample non-conducting. The action of scanning the preparation techniques to remove the liquid