Scanning Electron Microscopy

In order to measure the diameters of the smaller fibres, it was necessary to use a technique which allowed objects that were less than 200 nm to be viewed so scanning electron microscopy (SEM) was chosen. SEM uses electrons as a probe to image the top layer of a sample, with a depth that depends on the material of the sample and on the energy of the electrons, and can range from 1 nm to 1 μm. As opposed to other types of electron microscopies, such as TEM, SEM can rapidly image large areas of sample and is relatively easy to operate. It also uses a relatively low energy of electrons and therefore does typically not cause as much damage to a soft biomaterial as TEM. A Zeiss SUPRA 55-VP FEGSEM (field emission gun scanning electron microscope) is used for the following work.

In order to create a micrograph with SEM, a beam of primary electrons is directed towards the sample. These electrons interact with the specimen and cause other electrons to be emitted; these latter, so-called secondary electrons, are detected at a low angle (close to the surface) and used for creating an image of the sample. Secondary electrons are ejected from the valence band of the sample and carry information about the surface topography as their low energy does not allow them to escape from the sample if they have been generated more

than around 50 nm below the surface.4

It is also possible to use backscattered electrons which are higher in energy and detected at a higher angle (close to the electron source). They carry information about the topography and atomic number of the sample, and probe it to a larger depth.5

The beam of electrons is scanned over the sample in a raster fashion to cover all points in a selected area. The secondary electron detector records an intensity at each point in the raster scan. This intensity is input to a cathode-ray tube (CRT) whilst the scan is taking place which produces a digital image of the sample in real time.6

The fact that SEM uses electrons instead of visible light means that it has a resolution of about 10 nm. This is because the energy of electrons typically used in SEM corresponds to a wavelength which is much smaller than the wavelength of visible light. Equation 2.1 shows that wavelength is directly proportional to the resolution, so that shorter wavelength electrons allow smaller objects to be observed.

SEM is typically an ionising technique and it is thus necessary to coat non- conductive samples with a conductive layer and then to ground them before placing them in an SEM. This prevents sample charging which would distort the electrons trajectories and thus the resulting image. Graphite was used here to coat the FF fibres. This results in a 250 Å layer of amorphous conductive carbon layer deposited on the sample.

A 10 μL solution of FF fibres in water was deposited onto a silicon substrate with a pipette and this was left in a fume hood overnight so that all of the water evaporated. This dried sample was coated in amorphous carbon and placed into the SEM (initial attempts to image uncoated samples resulted in highly distorted images). For this work the samples were imaged using a primary electron beam energy of 2 keV. In general, higher electron energies produce better quality images, however 5 keV was found to damage the fibres and 2 keV was therefore used as the best compromise between image quality and sample preservation. The main drawback of SEM is that the sample must be dried, carbon coated, and then placed in a vacuum in order to be imaged. These conditions are far from the

standard conditions in which the fibres grow. This makes it impossible to look at any dynamic properties of the fibres with this technique.

2.1.2.1 Drying Effects

A further drawback of the necessity to work with dry samples is that the very act of drying a sample of fibres in solution can significantly alter its size distribution. In fact, as long as peptides are present in solution, removal of the solvent can cause a rapid and spatially inhomogeneous increase of the local supersaturation which leads to their further assembly into fibres, both adding to already existing fibres or creating new ones. The resulting sample is thus generally quite different from the starting sample and not representative of the fibre size and shape distribution as would have been observed under the standard conditions defined at the beginning of this chapter.

Several attempts were made to minimise or avoid these drying effects in order to use SEM to characterise the fibres. These methods all involved taking aliquots of the sample at different times during the growth process, followed by different attempts to remove the solution without evaporation. These methods included filtering and rapid solution pouring and are described in detail in Chapter 3. Another method used to avoid drying effects was cryo-SEM as described in the next subsection.

2.1.2.2 Cryo-Scanning Electron Microscopy

The main drawback of SEM is that it is impossible to observe a sample in ambient conditions. Whilst cryo-SEM does not completely overcome this problem, it does go some way to addressing it. Instead of drying the sample, a drop of it is frozen quickly in its solvent, typically with liquid nitrogen, and this frozen sample is placed in the SEM. This requires a special attachment chamber to a standard SEM. In this work the Zeiss SUPRA 55-VP FEGSEM was used with the Gatan Alto 2500 cryo-transfer system. After the sample has been frozen it is placed in this cryo-chamber and a knife which is inside the chamber is used to slice the drop of frozen sample. This exposes some of the fibres embedded in the frozen solvent and standard SEM can be used to image the exposed surface. The sample stage within the SEM is cooled in order to prevent sublimation.