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Cryo-Ultramicrotomy


Table 1 : Information of common LCs used in this article: names, molecular structures, LC types, and phases. Common name Full name


Molecular structure DSCG Disodium cromoglycate LC type


Lyotropic chromonic


Phase used in this article


Nematic


Sunset yellow FCF (SSY)


6-hydroxy-5-[(4-sulfophenyl) azo]-2-naphthalenesulfonic acid


Lyotropic chromonic


Nematic


5CB E7 CB7CB


4-Cyano-4’-pentylbiphenyl


Mixture of several cyanobiphenyls with long aliphatic tails


1”,7”-bis(4-cyanobiphenyl-4’- yl)heptane


Thermotropic Nematic


Thermotropic Cholesteric due to chiral doping


Thermotropic Twist bend nematic


vitrifi cation of the aqueous sample can be achieved [ 2 ]. A short section (< 5 mm) was cut from the capillary tube using a special cutter to provide a block of frozen specimen for cryo-ultramicrotomy. For “bulk” thermotropic LCs , a drop of sample a few hundred micrometers thick was applied to a copper or aluminum pin, followed by thermal treatment and plunge freezing ( Figure 2d ). Cryo-ultramicrotomy . Figures 2 c and 2 d also demonstrate how cryo-ultramicrotomy was performed on rapidly frozen “bulk” samples. In this study, we used a Leica UC7/FC7 cryo-ultramicrotome equipped with a microma- nipulator and a Crion anti-static device. T e cryo-chamber temperature (including knife and sample) should be below the glass transition temperature of the material and was normally between -120°C and -160°C. A cryo-trim diamond knife (Diatome) with a 45° cutting angle and a 20° side angle was used to trim the top of the block into a pyramid shape with a 50 µm height and a 50–200 µm width. Fine sectioning of electron-transparent slices was carried out using cryo-diamond knifes with 35° or 25° cutting angles. A slow cutting rate (typically 1 mm/sec or less), a small cutting width, and a thinner slice were used to achieve easier gliding of slices on the knife surface, which reduced damage. Continuous ribbons were obtained for all the samples described in this article, thanks to a modifi ed procedure demonstrated by Mr. Helmut Gnägi. As illustrated in Figures 2 c and 2 d, a wedge section of a Cu TEM grid was held by tweezers controlled by the micromanipulator. T e starting point of the ribbon was


2018 March • www.microscopy-today.com


attached to the Cu wedge by an eyelash tool. The attachment was enhanced by the “charge” function of the anti-static device. The manipulator was adjusted to accommodate the growing length of the ribbon during the sectioning process. When the ribbon was comfortably longer than 3 mm, a C-flat holey carbon-coated TEM grid was placed under the ribbon to collect the ribbon. The “charge” function of the anti-static device was used again to enhance adhesion. It should be mentioned that all tools need to be cooled to the cryogenic temperature before touching the ribbons. Cryo-TEM . The prepared cryo-TEM specimens were stored in liquid nitrogen. For cryo-TEM observation, the specimen was loaded onto a Gatan 626.DH cryo-holder. The cryo-holder with its liquid nitrogen dewar was then transferred into the TEM. Low-dose cryo-TEM observations were performed using an FEI Tecnai F20 TEM equipped with a Gatan Twin-blade anti-contaminator. A Gatan UltraScan 4K camera was used to record images [ 4 ]. During the TEM observation process, the specimen was kept below -170°C by liquid nitrogen in the dewar of the cryo-holder.


Results T in fi lm approach . Figure 3 shows a few examples from our recent eff ort to revisit the method of direct imaging of rapidly frozen thin fi lm specimens (see Figures 2 a and 2 b). Figure 3a shows the side and top views of the columnar aggregates in a novel lyotropic chromic LC of Ag-containing disk molecules [ 6 ]. T e top view of the aggregates (insert image) shows the nature of the nematic arrangement. In a thermotropic


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