Cryo-sectioning method

The determination of 3D structure by cryo-EM methods follows a common scheme for all macromolecules (Fig. 14.2). A more detailed discussion of the individual steps as applied to different classes of macromolecules appears in subsequent sections. Briefly, each specimen must be prepared in a relatively homogeneous, aqueous form (ID or 2D crystals or a suspension of single particles in a limited... 

For purely biological studies of embryogenesis, both paraffin section and cry-section are good options for well-preserved tissue and organ samples. However, for liquid-crystal functional studies during embryo development and pathological events, cryo- or frozen-section are preferred over paraffin sections, as the cryo/frozen methods preserve more of the sample s original characteristics than the more chemically intensive paraffin preservation method. If possible, fresh sample smear-slides are also preferred. [Xu MM et al 2009, Xu XH et al 2009]. 

The goal of our investigations was to characterise the morphology of the sample, and to determine the size and location of the PTFE and silicone oil phases by different methods [46,47], For phase characterization using Raman microscopy, no special sample preparation was necessary. For FTIR imaging, microtomed sections (5 pm in thickness) had to be prepared by cutting the sample with a diamond knife at — 80°C ("cryo-microtomy") to prevent smearing and to obtain flat surfaces. 

Microemulsion microstructures are best imaged by cryo-TEM. A description of sample preparation methods with a view to obtaining direct and artifact-free images is described in this section as it is the key to microscopy. 

Ultramicrotomy (including cryo-ultramicrotomy) is a standard method for the preparation of ultrathin or semithin sections, as well as very flat surfaces for various microscopic investigations. Improvements in preparation techniques over the last few decades have demonstrated that thin sections of different materials that are free from artifacts can be successfully prepared for EM investigations. Therefore, successful sectioning now depends primarily on the experience of the experimentalist rather than on the instrumentation used [14]. 

Cryo-SEM and cryo-TEM are the methods that have been developed for study of polymers at low temperatures, and these will be discussed in this section. Low temperature stages for AFM are just becoming available. There is some published work on PDES (polydiethylsi-loxane) materials obtained in an AFM cooled to -50°C using thermoelectric cooling [588]. 

It is well known that the particle shape, size, and distribution of a latex or emulsion control the properties and end-use applications. Many types of latex are manufactured with a controlled and sometimes monodisperse distribution of particle sizes. These polymer liquids are wet and sticky, making specimen preparation for microscopy very difficult. Because particle size and shape are so important to properties, the preparation must focus on not changing the particles as found in the fluid state. Preparation includes simple methods (see Section 4.1) such as dropping a solution onto a specimen holder, staining/fixation (see Section 4.4), microtomy (see Section 4.3), and special cryo methods (see Section 4.9). All microscopy techniques can be used for these studies. This section is meant to provide a brief survey of the types of microscopy applications that have been found useful in the evaluation of emulsions, latexes, and their use as coatings and adhesives. 

The distribution of components in BHJ thin-films normal to the surface of the film is also critical in defining the performance of the material. In the simplest extreme, if there is a preferential segregation of one eomponent to an electrode interface, device performance can be poor even with an idealized morphology in the remaining part of the BHJ active layer. Given that charge transfer and collection occur at the electrode, transport to these interfaces requires transport normal to the surface of the film. Normally, the BHJ layer is 100-200 nm in thickness. Consequently, methods are needed to assess composition profiles normal to the film surface or teehniques must be used to examine cross-sections normal to the film surfaee. In the latter case, cryo-microtoming or focused ion beam (FIB) are required. 

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