Cryo-Ultramicrotomy of Complex Molecular Fluids

Min Gao Liquid Crystal Institute , Kent State University , 1425 Leſt on Esplanade , Kent , Ohio 44242

Abstract: Cryogenic transmission electron microscopy (cryo-TEM) has become a powerful tool for probing the structures of soft- matter materials at nanometer or sub-nanometer scale. For many complex molecular fl uids (CMFs), the limiting factor in such studies is the preparation of a cryo-TEM specimen that represents the native structure. Plunge freezing of electron-transparent thin fi lms has been widely used, but it is often infl uenced or even dominated by surface effects and mechanical disturbances of the CMF. In this article, we use liquid crystals as model CMF systems and present the general procedures and typical results of cryo-ultramicrotomy of rapidly frozen “bulk” samples.

Keywords: Cryo-TEM, cryo-ultramicrotomy, complex molecular fl uids, liquid crystals, high-pressure freezing

Introduction Complex molecular fl uids (CMFs), such as crude oil, surfactants, and liquid crystals, have tremendous impacts on the modern world and are the foundation of several major industries. Unlike simpler liquids, CMFs normally exhibit complex structures (for example, micelles, vesicles, and liquid crystal mesophases) in addition to basic molecular liquids and crystal phases. Figure 1 shows two main types of liquid crystals (LCs) between true liquids and true solids: nematic mesophases (with orientational order only) and smectic mesophases (with orientational order and 1- or 2-D positional order). Organic LCs include thermotropic (typically rod-like molecules with phase transitions driven by temperature) and lyotropic LCs (mixtures of organic molecules and a solvent with ordering transitions governed mainly by concentration) [ 1 ]. Because of their complicated structures and the lack of eff ective nanoscale structural probes for CMFs, detailed understanding of CMF behavior at the molecular level is oſt en surprisingly limited. Cryogenic transmission electron microscopy (cryo-TEM), recently in the Nobel Prize spotlight for its application in structural biology, has evolved into an essential technique for direct visualization of soſt -matter materials at sub-nanometer scale. In cryo-TEM studies, native (oſt en dynamic) structures are quenched into static structures using rapid freezing and observed with low-dose electron beams at cryogenic temperatures. To prepare a cryo-TEM specimen, plunge freezing of a partially electron- transparent thin fi lm is normally the “go-to” technique, as shown in Figure 2a [ 2 ]. However, it has been shown that mechanical disturbance (for example, shear force) during the blotting process to remove excess


liquid can alter sensitive CMF structures dramatically. For example, wormlike micelles could be modifi ed into individual micelle particles [ 3 ]. In addition, many CMFs including LCs are sensitive to surface and interface eff ects, making it challenging to preserve the native structure in electron-transparent thin fi lms [ 4 ].

In this article, we focus on an alternative “bulk” approach where much thicker samples are rapidly frozen and sectioned by cryo-ultramicrotomy to obtain electron-transparent thin fi lms. We use liquid crystals as a model CMF system and present the general procedures for applying cryo-ultramicrotomy in a variety of LC phases. Some of the better-known applications of cryo-ultramicrotomy can be found in rubber and polymer research and the Tokuyasu immunolabeling technique for sucrose-cryoprotected biological tissues. Very diff erent from those semi-solid materials, LCs and other CMFs are aqueous solutions (lyotropic LCs) or loosely bound molecules (thermo- tropic LCs). Cryo-sectioning of these super-cooled viscous fl uids is similar to that for vitrifi ed aqueous biological samples in the challenging practice of cryo-electron microscopy of vitreous sections (CEMOVIS). T e CEMOVIS method combines high-pressure freezing, cryo-ultramicrotomy, and cryo-TEM and makes possible the study of biological samples (such as tissues, cells, and bacteria) in their fully hydrated states [ 2 ]. Cryoprotectants are oſt en added to improve vitrifi - cation and sectioning quality. High-pressure freezing has also been employed to vitrify lyotropic LCs so the present methods for this type of LC can be considered a part of CEMOVIS. Lyotropic LCs are normally homogeneous phases with high water contents, and additional cryoprotectants can have a greater impact on their structures than on biological samples.

Figure 1 : A simplifi ed picture comparing nematic and smectic liquid crystal mesophases to molecular liquid and crystal phases. Low-temperature and high molecular concentration tend to enhance the degree of order in thermo- tropic and lyotropic liquid crystals, respectively.

doi: 10.1017/S1551929517001390 • 2018 March

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