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Cryo-Ultramicrotomy


Figure 7 : Comparative cryo-TEM images of LC-nanoparticle composites with gold nanoparticles incorporated into 4-Cyano-4’-pentylbiphenyl (5CB). (a) Thin fi lm approach (plunge freezing using the FEI Vitrobot) imaged in bright-fi eld TEM mode. (b) Bulk approach (plunge-freezing and cryo-sectioning). Annular dark-fi eld (Z-contrast) STEM imaging improves the visibility of the nanoparticles within the liquid crystal. The contrast variation in the background is corresponding to the imperfection caused by cryo-ultramicrotomy.


Discussion


Currently, there are two main challenges facing the recommended “bulk” approach specimen preparation method for cryo-TEM of LCs and other CMFs. First, the freezing of “bulk” samples may not be straightforward. T e slower cooling rate further away from the sample surface may introduce undesired phase transitions, especially in thermotropic LCs.


2018 March • www.microscopy-today.com


T is is a lesser problem for lyotropic LCs since high-pressure freezing can be used. In addition to plunge freezing, other techniques are available to achieve faster cooling rates, for example, slam freezing and jet freezing [ 2 ]. But plunge freezing is the most convenient one, and it is oſt en suffi cient for thermotropic LCs because of the slower diff usion of the large molecules compared to water-containing solutions, especially for those samples possessing super-cooling properties. Second, the cryo-sectioning of frozen LCs or CMFs in general can be challenging at this point. T e quality of the sectioned cryo-TEM specimens may depend to a large degree on the skill and experience of the operator. Cryo-ultramicrotomy of frozen CMFs is a dry-cutting process of a high-viscosity fl uid using a sharp diamond knife. T e sectioned thin slices need to glide on the knife surface. Also, the frozen fl uids have no strong internal bonding and can fl ow and deform depending on external force. During cutting, the knife edge creates high local pressure, which introduces inevitable compression along the sectioning direction and thickness increases. Crevasses occur when the fracture limit of the sample is exceeded. In addition, many inhomogeneities exist including irregularities and damage at the knife edge, imperfections and irregular friction of the knife surface, and inhomogeneous sample, just to name a few [ 2 ]. T ese factors can contribute to various image artifacts (for example, knife marks and varying section thickness). As a result, the imaging quality from the sectioned specimens is normally not as good as that from the thin fi lm approach. An optimized cryo-ultramicrtomy condition is a key for high-resolution imaging of cryo-sectioned LC specimens. Recent instrumentation developments in several areas present a promising future for cryo-sectioning. For example, single/double manipulators [ 11 ], the anti-charging device, and improved diamond knives have helped the quality of cryo-ultramicrotomy. In addition, focused ion beam sectioning operating at cryo-temperatures (cryo-FIB) may be a promising alternative to cryo-ultramicrotomy. Cryo-FIB advantages may include less sectioning damage, more fl exibility in sectioning direction, more accurate access to the diff erent depths of the frozen sample, and better utilization of the surface layer [ 12 ]. We also demonstrated here that the thin fi lm and bulk approaches to cryo specimen preparation can be comple- mentary, as can cryo-TEM and replica freeze-fracture TEM. T e advantages provided by the thin fi lm approach (straight- forward freezing, simple geometry, high imaging quality, etc.) and freeze fracture TEM (surface sensitivity, etc.) can be very useful when used properly. A combination of the above techniques is recommended for challenging systems.


Conclusion


In this article we present procedures and representative results on the “bulk” approach for studying nanoscale structures in a range of liquid crystals. T e “bulk” approach, combining rapid freezing of complex molecular fl uids, cryo-sectioning, and cryo-TEM, can eff ectively minimize the surface/interface eff ects that have been a major limitation of the more convenient thin fi lm approach cryo-TEM. T us, the bulk approach reveals more precisely the native structure. We believe that the bulk approach can become a powerful alternative to the thin fi lm


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