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Direct Observation of Liquid Crystals Using Cryo-TEM: Specimen Preparation and Low-Dose Imaging MIN GAO, 1 * YOUNG-KI KIM, 1 CUIYU ZHANG, 1 VOLODYMYR BORSHCH, 1 SHUANG ZHOU, 1 HEUNG-SHIK PARK, 1 ANTAL J AKLI, 1 OLEG D. LAVRENTOVICH, 1 MARIA-GABRIELA TAMBA, 2 ALEXANDRA KOHLMEIER, 2 GEORG H. MEHL, 2 WOLFGANG WEISSFLOG, 3 DANIEL STUDER, 4 BENO ^ IT ZUBER, 4 HELMUT GN AGI, 5 AND FANG LIN 6 1 Liquid Crystal Institute and Chemical Physics Interdisciplinary Program, Kent State University, Kent, Ohio 44242 2 Department of Chemistry, University of Hull, Hull, HU6 7RX, United Kingdom 3 Department of Chemistry and Physical Chemistry, Martin Luther University Halle-Wittenberg, von-Danckelmann-Platz 4, Halle 06120, Germany 4 Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000 Bern 9, Switzerland 5 Diatome Ltd Switzerland, P.O. Box 557, CH-2501 Biel, Switzerland 6 College of Science, South China Agricultural University, Guangzhou, 510642, China KEY WORDS freeze fracture; high-pressure freezing; CEMOVIS; thermotropic; lyotropic ABSTRACT Liquid crystals (LCs) represent a challenging group of materials for direct trans- mission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset ena- bling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present com- parative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understand- ing of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solu- tion to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. V C 2014 Wiley Periodicals, Inc. INTRODUCTION Transmission electron microscopy (TEM) techniques are under fast development in terms of, e.g., resolving power (Meyer et al., 2008), temporal resolution (Kim et al., 2008), and in situ environmental capability (Gai and Boyes, 2009). However, for many real-world prob- lems, the outputs (for example, imaging resolution) are often limited by the material properties rather than by the microscope performance. On the other hand, the technical developments in microscopy-related fields enable us to revisit some of the earlier challenging problems and get much improved results. In this paper, we report some of the initial results of our on- going efforts to re-pursue high resolution direct TEM imaging of liquid crystals (LCs), a group of materials having tremendous impact (e.g., liquid crystal display, the so-called LCD) but imposing great challenges for direct TEM observation. LCs uniquely combine order and mobility, and are generally described as intermediate states (meso- phases) of matter between crystalline solid (possessing both orientational and 3D positional orders) and liquid (with neither orientational nor positional orders) (Col- lings, 2002; De Gennes and Prost, 1995). Organic LCs can be divided into thermotropic and lyotropic LCs, with the phase transition (change in the amount of order) mainly driven by temperature in the former, and by concentration or percentage of the added sol- vent (most often, water) in the latter. A typical single component thermotropic LC consists of molecules (mesogens) with, for example, rigid rod-like or some- times disk-like middle part and attached flexible ends. Thermotropic LC materials may exhibit one or more mesophases (most commonly nematics and smectics) between the high-temperature isotropic liquid and the low-temperature crystalline phases. Simply speaking, *Correspondence to: Min Gao, Liquid Crystal Institute, Kent State University, 1425 University Esplanade, Kent, Ohio 44242, USA. E-mail: [email protected] REVIEW EDITOR: Prof. Jian-Min (Jim) Zuo Received 13 February 2014; accepted in revised form 23 June 2014 DOI 10.1002/jemt.22397 Published online 5 July 2014 in Wiley Online Library (wileyonlinelibrary.com). V V C 2014 WILEY PERIODICALS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 77:754–772 (2014)
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  • Direct Observation of Liquid Crystals Using Cryo-TEM:Specimen Preparation and Low-Dose ImagingMIN GAO,1* YOUNG-KI KIM,1 CUIYU ZHANG,1 VOLODYMYR BORSHCH,1 SHUANG ZHOU,1

    HEUNG-SHIK PARK,1 ANTAL J�AKLI,1 OLEG D. LAVRENTOVICH,1 MARIA-GABRIELA TAMBA,2

    ALEXANDRA KOHLMEIER,2 GEORG H. MEHL,2 WOLFGANG WEISSFLOG,3 DANIEL STUDER,4

    BENOÎT ZUBER,4 HELMUT GN€AGI,5 AND FANG LIN61Liquid Crystal Institute and Chemical Physics Interdisciplinary Program, Kent State University, Kent, Ohio 442422Department of Chemistry, University of Hull, Hull, HU6 7RX, United Kingdom3Department of Chemistry and Physical Chemistry, Martin Luther University Halle-Wittenberg, von-Danckelmann-Platz 4, Halle06120, Germany4Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000 Bern 9, Switzerland5Diatome Ltd Switzerland, P.O. Box 557, CH-2501 Biel, Switzerland6College of Science, South China Agricultural University, Guangzhou, 510642, China

    KEY WORDS freeze fracture; high-pressure freezing; CEMOVIS; thermotropic; lyotropic

    ABSTRACT Liquid crystals (LCs) represent a challenging group of materials for direct trans-mission electron microscopy (TEM) studies due to the complications in specimen preparationand the severe radiation damage. In this paper, we summarize a series of specimen preparationmethods, including thin film and cryo-sectioning approaches, as a comprehensive toolset ena-bling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present com-parative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic andlyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced,e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioningof lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEMtechniques. The significance of subnanometer resolution cryo-TEM observation is demonstratedin a few important issues in LC studies, including providing direct evidences for the existence ofnanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understand-ing of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropicchromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggestingthat TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solu-tion to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res.Tech. 77:754–772, 2014. VC 2014 Wiley Periodicals, Inc.

    INTRODUCTION

    Transmission electron microscopy (TEM) techniquesare under fast development in terms of, e.g., resolvingpower (Meyer et al., 2008), temporal resolution (Kimet al., 2008), and in situ environmental capability (Gaiand Boyes, 2009). However, for many real-world prob-lems, the outputs (for example, imaging resolution) areoften limited by the material properties rather than bythe microscope performance. On the other hand, thetechnical developments in microscopy-related fieldsenable us to revisit some of the earlier challengingproblems and get much improved results. In thispaper, we report some of the initial results of our on-going efforts to re-pursue high resolution direct TEMimaging of liquid crystals (LCs), a group of materialshaving tremendous impact (e.g., liquid crystal display,the so-called LCD) but imposing great challenges fordirect TEM observation.

    LCs uniquely combine order and mobility, and aregenerally described as intermediate states (meso-phases) of matter between crystalline solid (possessing

    both orientational and 3D positional orders) and liquid(with neither orientational nor positional orders) (Col-lings, 2002; De Gennes and Prost, 1995). Organic LCscan be divided into thermotropic and lyotropic LCs,with the phase transition (change in the amount oforder) mainly driven by temperature in the former,and by concentration or percentage of the added sol-vent (most often, water) in the latter. A typical singlecomponent thermotropic LC consists of molecules(mesogens) with, for example, rigid rod-like or some-times disk-like middle part and attached flexible ends.Thermotropic LC materials may exhibit one or moremesophases (most commonly nematics and smectics)between the high-temperature isotropic liquid and thelow-temperature crystalline phases. Simply speaking,

    *Correspondence to: Min Gao, Liquid Crystal Institute, Kent State University,1425 University Esplanade, Kent, Ohio 44242, USA. E-mail: [email protected]

    REVIEW EDITOR: Prof. Jian-Min (Jim) Zuo

    Received 13 February 2014; accepted in revised form 23 June 2014

    DOI 10.1002/jemt.22397Published online 5 July 2014 in Wiley Online Library (wileyonlinelibrary.com).

    VVC 2014 WILEY PERIODICALS, INC.

    MICROSCOPY RESEARCH AND TECHNIQUE 77:754–772 (2014)

  • a nematic phase has only orientational order but nolong-range positional order. In the best known uniaxialnematics, the randomly positioned molecules alignfavorably along a single axis, the so-called director n̂.In addition to the orientational order, a smectic phasealso has a degree of positional order, as the moleculestend to form layers stacked on top of each other. Thereis no further positional order inside each layer for thetwo common smectic phases: smectic A (SmA), inwhich the rod-like molecules are parallel to the layernormal, and smectic C (SmC), in which the moleculesare tilted with respect to the layer normal. While for asmectic B (SmB) phase, the molecules, orienting onaverage along the layer normal, are positioned in ahexagonal lattice within the layers. Details on thestructure of other smectic and crystalline-like meso-phases can be found elsewhere (De Gennes and Prost,1995).

    Lyotropic LCs are composed of mixed LC moleculesand solvent. The most well-known lyotropic LCs areformed by amphiphiles, i.e., molecules with one hydro-philic “head” and one or two hydrophobic ends. Repre-sentative examples are soaps, phospholipids, andsurfactants. Driven by hydrophobic/hydrophilic inter-actions, the amphiphilic molecules in solvent (usuallywater) aggregate into vesicles and micelles. At suffi-cient concentrations, the aggregates of amphiphilicmolecules of different shapes pack into mesophasessuch as lamellar, hexagonal columnar and cubicphases depending mainly on the concentration andproperties of the hydrophobic and hydrophilic molecu-lar parts. A distinct but broad family of lyotropic LCsis formed by chromonic LCs, consisting of mesogenswith disk-like aromatic central core and ionic outergroups (Park and Lavrentovich, 2012). While in water,the molecules self-assemble into columnar aggregatesby face-to-face stacking that minimizes the contactarea with water. Mainly two LC phases can be formedby these self-assembled columnar aggregates: a low-concentration nematic phase and a high-concentrationhexagonal columnar phase, often called “M phase”(Lydon, 2010). Since the molecules in aggregates arebound together by weak non-covalent interactions, thelength of the aggregates depends strongly on tempera-ture. Thus the phase diagrams of chromonic LCs,unlike their amphiphilic lyotropic counterparts, arevery sensitive to the temperature (Park and Lavrento-vich, 2012).

    The understanding of detailed LC behaviors at themolecular level is surprisingly limited, which can bepartly attributed to their uniquely complicated struc-ture and the lack of effective structural probe at nano-meter and subnanometer scale. Beyond the classicpolarized light microscopy (PLM) characterizing struc-tures at a scale of micrometers, x-ray diffraction,including synchrotron small-angle x-ray scattering(SAXS), has been the most widely used nanoscalestructural characterization tool in LCs (Hong et al.,2010), targeting mainly ordered structures with peri-ods less than tens of nanometers. Nanoscale imagingtechniques, e.g., TEM, scanning electron microscopy(SEM) (Rizwan et al., 2007), scanning tunnelingmicroscopy (Frommer, 1992), and atomic force micros-copy (Yashima, 2010), have also been used, thoughtheir applications so far have been relatively limited.

    Among the imaging techniques, a replica TEM tech-nique, namely freeze fracture TEM (FFTEM), appliedto LCs since 1970’s (Costello et al., 1984; Kl�eman et al.,1977; Lydon and Robinson, 1972), has attracted contin-uous interests by revealing the internal structures of avariety of LC materials (Borshch et al., 2013; Chenet al., 2013; Hough et al., 2009).

    Two major challenges exist for the successful appli-cation of direct TEM imaging in LC studies: preparingTEM specimens with preserved native structure andminimizing radiation damage during TEM observa-tion. The LC materials are similar to organic liquids,in which the small molecules (typically a few nano-meters long) are moving rather freely and spend only alimited time at the preferred orientation. The forceskeeping the orientational order are very weak, whichmakes it easy to use electrical or magnetic field tomanipulate the overall director for practical applica-tions, but also leads to a relatively high sensitivity toelectron beam as compared even to typical biomateri-als. For TEM specimen preparation, LCs also posesome unique challenges in addition to the problemsshared by many non-solid materials. At the currentstage, it is common to use cryo-TEM to observe plunge-frozen thin films of fluid-like materials that are elec-tron transparent. However, the molecular alignmentin LCs is highly sensitive to the presence of surfacesand interfaces. The reason is that the surfaces andinterfaces introduce aligning forces acting on the LCbulk, thanks to the anisotropy of surface interaction(the so-called surface anchoring phenomenon)(J�erôme, 1991). As a result, thin specimens may showstructures strongly influenced by the surface forces.Some surface-induced structures can be very differentfrom the bulk LC structures, as shown in the examplesof the so-called helical nanofilament phase in contactwith glass substrate (Chen et al., 2012) and thenematic LC that develops smectic layering near thesurface (Ruths et al., 1996). In addition, the surfaceanchoring may set the molecular orientation in a direc-tion (for example, parallel to the electron beam) that isdifficult for TEM observation. On the other hand, tofreeze a thick sample with minimized influence of thesurface forces is often not practical, because of theinsufficient cooling rate. The frozen thick samples mayfail to preserve the native structure, which is espe-cially true for lyotropic LCs with high water contentand some high-temperature thermotropic phases.

    Due to the above challenges, despite the promisingearly direct TEM imaging results (Bunning et al.,1994; Gilli et al., 1991; Hara et al., 1988; Voigt-Martinand Durst, 1987; Voigt-Martin et al., 1992), it is theindirect technique of FFTEM that remains to be adominant high-resolution imaging tool for LC bulkstructures nowadays. In FFTEM, a frozen LC sampleis first fractured at low temperature in vacuum, andthen the fractured surface is replicated by a thin depo-sition of heavy metal at an angle to create shadows ofthe surface topography, followed by a continuous car-bon supporting layer deposited from the top. The reso-lution of the FFTEM is largely determined by theparticle size of the deposited heavy metal, which isnormally a few nanometers. Although FFTEM effec-tively avoids radiation damage by using replica speci-mens, it disables the use of many TEM techniques

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  • (e.g., diffraction and spectroscopy), and has a very lim-ited potential towards sub-nanometer imaging whichis essential to understand LC behaviors (phase transi-tion, defect, interfaces, etc.) at the molecular scale.

    In this paper, we demonstrate a series of specimenpreparation routines for LCs, aiming to set up an eas-ily accessible toolbox for TEM imaging especiallydirect cryo-TEM examination of the majority of ther-motropic and lyotropic LC phases. In addition to adopt-

    ing and modifying a few of the previous practices(Kostko et al., 2005; Pierron et al., 1995), some newconcepts are introduced, e.g., suspended thermotropicLC thin films, cryo-sectioning of high pressure frozenlyotropic LCs, and complementary combination ofdirect cryo-TEM and replica FFTEM. For sub-nanometer resolution imaging, we demonstrate that awidely available cryo-TEM, a high sensitivity CCDcamera and a modified low-dose imaging procedurehave made a ready combination for many challengingLC materials. We present examples of applying theabove techniques to reveal the presence of nanoscalesmectic clusters in bent-core nematic LCs, the exis-tence of twist-bend nematic mesophase with a complexdirector structure that follows the geometry of anoblique helicoid with a nanoscale pitch, and nematicarrangement of the elongated aggregates in the lyo-tropic chromonic LCs. Direct TEM imaging opens thedoor for applying a variety of TEM techniques beyondlow-dose imaging, which makes TEM a promisingmolecular scale structural probe for LC and other soft-matter materials.

    MATERIALS AND METHODSLC Materials

    Figure 1 shows the chemical structure of the LCmolecules discussed in this paper. The formal namesand their short forms are both given. We report on themicrostructure of three thermotropic (namely, 3RBC-S, 3RBC-N, and CB7CB) and two lyotropic chromonic(DSCG and SSY) LCs. The two three-ring bent-core(3RBC) LCs, are formed by banana-like molecules, inwhich a bent rigid core has two aliphatic flexiblechains attached to its ends (Weissflog et al., 2012). The3RBC-S forms a SmA phase, while the 3RBC-N formsa nematic phase. In the case of dimeric thermotropicmesogen CB7CB, the molecule represents two rigidrod-like segments connected by a flexible central ali-phatic bridge (Panov et al., 2010). As representativesof chromonic LCs, we used the two most widely studiedmesogens (Park and Lavrentovich, 2012): disodiumcromoglycate (DSCG), known also as an anti-asthmatic drug, and Sunset Yellow FCF (SSY), a fooddye. The DSCG samples used here have concentrationsof 6.2 wt% (0.129 mol/kg) and 15 wt% (0.344 mol/kg) inwater, determined by PLM to be in isotropic andnematic phases at room temperature, respectively(Kim et al., 2013). The nematic SSY sample is 30.7wt% (0.98 mol/kg) with 64.8% water and 4.5% MgSO4(Zhou et al., 2012). In addition, we show results on Aunanoparticle doped 5CB.

    TEM Specimen Preparation Procedures

    Figure 2 is a schematic overview showing the basicroutines used in this study to prepare TEM specimensof thermotropic (Figs. 2a–2c) and lyotropic (Figs. 2d–2f) LCs for cryo-TEM and FFTEM. Rapid freezing isused in all the routines to quench the flowing LC mate-rials, making the static structure inside a TEM speci-men basically a snapshot of the LC material with bothorientational and positional fluctuations. Here we cat-egorize the techniques into “thin film” approach and“bulk” approach based on the form of the pre-freezingsample. In the former, the LC material or at least partof it is already electron transparent and can be

    Fig. 1. Molecular structures and names (full and short) of the LCmaterials used in this article.

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  • Fig. 2. TEM specimen preparation routines for thermotropic LCs(TLC) (a–c) and lyotropic LCs (LLC) (d–f). (a) Plunge freezing of sup-ported and suspended TLC thin films for cryo-TEM. (b) Cryo-sectioningof plunge frozen “bulk” TLC for cryo-TEM. (c) Freeze fracture of plungefrozen “bulk” TLC for FFTEM. (d) Plunge freezing of LLC thin film for

    cryo-TEM. (e) Vitreous sectioning of high pressure frozen LLC for cryo-TEM. (f) Freeze fracture of frozen (plunge freezing or high-pressurefreezing) LLC for replica TEM. The scale bars in the schematics roughlydemonstrate the feature sizes. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

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  • transferred directly into the TEM after the freezingprocess. While for the “bulk” approach, much thicker(a few microns to hundreds of microns) material is firstfrozen and then goes through further processing toobtain electron transparent TEM specimen. Thedetailed procedures of the methods used in this studyare described as follows.

    Thin Film Approach: Plunge Freezing of Ther-motropic and Lyotropic LCs. For thermotropicLCs, we employed a simple but effective procedure toobtain LC thin films either suspended or supported bythin carbon film. As shown in Figure 2a, the LC mate-rials were first dissolved in an evaporable solvent(most often chloroform in this study) to make a �0.1–0.3% solution. A few microliters of the solution wereput on carbon (continuous or holey) coated TEM grids.The grids are normally treated with oxygen plasma ina Fischione 1020 plasma cleaner using 25% oxygenand 75% Ar mixture gas. LC thin films were depositedon the carbon films after the solvent evaporates, whilesuspended films were obtained in the holes of holeycarbon films. The thin films (together with the TEMgrids) then underwent thermal treatment to achievethe desired phase, followed by a fast plunge freezinginto liquid ethane or liquid nitrogen to preserve thestructure of the desired mesophase. The thermal treat-ment normally includes heating to the isotropic phase,and cooling down to the target temperature. To achieverepeatable results, enough time should be allowed forthe specimen to stabilize at each temperature and dur-ing the heating/cooling process. The thickness of thefilms can be controlled by the total volume of the LCsolution put on the grids and the concentration of thesolution, but normally a large variation in film thick-ness can be seen within the same TEM specimen. It isalso recommended to check the LC thin films usingPLM with a heating stage to make sure that the thinfilms still exhibit the basic characteristics of the LCmaterials, especially for multi-component materials.

    For lyotropic LCs with a high percentage of water(>80%), it is a straightforward process to prepare thinfilm cryo-TEM specimens using plunge freezing due totheir relatively low viscosity (Fig. 2d). In this study, weused a FEI Mark-IV Vitrobot to plunge-freeze the lyo-tropic samples at room temperature and �100%humidity. Less than 3 ll of the lyotropic LC solutionwas put on a lacey carbon coated TEM grid and blottedby two filter papers, followed by plunge vitrification inliquid ethane.

    Bulk Approach: Cryo-Sectioning of Thermo-tropic and Lyotropic LCs. In this study, cryo-sectioning was utilized to prepare TEM specimensfrom frozen LC materials (CB7CB, DSCG, and SSY).As shown in Figures 2b and 2e, the thermotropic andlyotropic LCs were first quenched using plunge freez-ing (in liquid nitrogen or ethane) and high-pressurefreezing (Leica EM PACT2), respectively. The followingcryo-sectioning was implemented at 2150�C or2160�C using a Diatome cryo 25� diamond knifemounted on Leica UC7/FC7 cryo-ultramicrotome. Thenominal slice thickness was set to 50–80 nm. The sec-tions were transferred onto holey lacey carbon coatedcopper grids. In the high-pressure freezing of lyotropicLCs, the LC solution was filled into copper capillarytubes of �300 lm in inner-diameter. A high pressure

    (�2,000 bar) was applied at the two ends of the tube todramatically slow down the crystallization of water.Immediately after the pressure application, liquidnitrogen was injected onto the tube to freeze the sam-ple (Studer et al., 2001). For the SSY sample contain-ing 64.8% water, no cryoprotectant was used in thevitrification. While for the DSCG samples with 85%and 93.8% water, 10–20% of dextran (Mr �70k) wasadded to vitrify the solutions successfully.

    Bulk Approach: Replica FFTEM. In this study,we also employ FFTEM to study thermotropic CB7CBand lyotropic DSCG. For CB7CB, the LC material wasput between two copper planchettes (Fig. 2c). Thesandwich structure was then stabilized at 125�C (iso-tropic phase), and cooled down to 95�C to obtain thelow-temperature twist-bend nematic phase (Borshchet al., 2013). After quenching in liquid nitrogen, theassembly was quickly transferred into the vacuumchamber of a freeze-fracture apparatus (BalTecBAF060) with the sample stage temperature set at2140�C. Inside the chamber, a built-in microtome wasused to break the assembly and expose the fracturesurface. �4 nm thick Pt/C was then deposited onto thefractured surface at a 45o angle to create shadowing ofthe surface morphology, followed by a �20 nm thick Cdeposition from the top to form a continuous support-ing film. The samples were then warmed up andremoved from the freeze fracture chamber. The LCmaterial was dissolved in chloroform, while the replicafilms were collected and placed onto carbon coatedTEM grids. For DSCG, the LC solution was vitrifiedusing high-pressure freezing (Fig. 2f), and fractured at2160�C. In Figure 2f, we also show schematically thewidely used plunge freezing routine to prepareFFTEM specimen from lyotropic LCs, in which a thinlayer of the LC solution (a few microns thick) is placedbetween two copper sheets.

    In our practice, complementary use of the above rou-tines has been applied to both thermotropic and lyo-tropic LCs, which helps towards comprehensiveunderstanding of the complicated LC behaviors. Inthis paper, we report results of combined thin filmplunge freezing, bulk cryo-sectioning, and bulkFFTEM, on two of the samples (CB7CB and DSCG).

    TEM Observation and a Modified Cryo-TEMLow-Dose Procedure

    TEM results shown in this paper were obtainedusing a versatile FEI Tecnai F20 microscope (200 kV)equipped with scanning TEM unit, energy-dispersivex-ray spectrometer (EDS), Gatan imaging filter, anti-contaminator, and low-dose operation mode. A Gatan626.DH cryo-holder was used to keep the specimentemperature

  • 1st condenser lens) even for high magnification(�70K–90K in our study). The searching and focusingof the area of interest (AOI) were carried out quickly atthe same magnification as the final exposure, but at amuch lower beam intensity/area controlled directly by“spot size”. The low-magnification searching was onlyused to locate regions in the LC films with suitablethickness. To minimize radiation damage, the beamwas always blanked when the specimen was notimaged. Compared to the dose level used for normalbiomaterials (10–25 e21/Å2), a much smaller dose levelwas normally required for thermotropic LCs. For someextremely sensitive materials, a dose of 0.2 e2/Å2 wasoften used for the rapid searching, and 2 e2/Å2 for theimage acquisition. The movement of specimen andunderfocus can help the searching of AOIs at low dose.It should be also pointed out that some LC materialsare relatively more robust under electron beam thanothers, and a higher dose can be applied to improvethe signal/noise ratio. On the other hand, the standardlow-dose procedure works well for lyotropic LCs whichnormally have uniform structure and have comparablebeam sensitivity to aqueous biological samples.

    To study the influence of surface effect and filmthickness, low-loss electron energy-loss spectroscopy(EELS) was used to determine the inelastic mean-free-path (MFP) and the thickness of the LC thin films(Egerton, 2011). The MFPs measured from severalthermotropic LCs materials range between 130 nmand 150 nm.

    RESULTS AND DISCUSSIONSubnanometer Resolution Imaging of

    Thermotropic and Lyotropic LCs Using ThinFilm Approach

    For a beam sensitive material, the achievable reso-lution in a given high-performance TEM is mainly lim-ited by statistical noise (depending on the electrondose level) and the property of the image recordingdevice. Our measurements (not shown) on the GatanUltrascan 4000 CCD camera (Zhang et al., 2003) usedin this study indicate that a dose rate of �10 e2/pixelor even less can allow an imaging resolution of 2–3 pix-els (considering the Nyquist theorem for 2-dimensional imaging). For example, at �10 e2/pixeland a magnification of 285K (binning 2, 0.78 Å/pixel),the 2.04 Å Au (200) lattice can be resolved, which is 1.3times the Nyquist frequency spacing (1.56 Å). Usingthis as a rough guidance, we can estimate that for ourroutinely used imaging condition (a magnification of71K at a binning of 2 for the CCD, leading to a pixelsize of 3.3 Å /pixel) and the minimum dose of 2 e2/ Å2

    (�22 e2/pixel without the specimen), a subnanometerresolution can be achieved for thin specimens. Ahigher resolution and “nice” images to bare eyes (withbetter signal/noise ratio) can be obtained for thosemore robust samples to which higher dose can beapplied. It should be pointed out that the results dem-onstrated in this paper were taken mostly from materi-als that can stand a higher dose.

    We have employed such “high resolution” directimaging to study the nanoscale structure of the two3RBC thermotropic LCs (Fig. 1), revealing the firstdirect evidence of smectic nanoclusters in the nematicphase of 3RBC-N (Zhang et al., 2012). The two mole-

    cules, 3RBC-S and 3RBC-N, have similar structureand length with rigid aromatic cores and flexiblehydrocarbon tails. 3RBC-S has a macroscopic SmAmesophase in the temperature range between 66.5�Cand 118.6�C. Figure 3a is a cryo-TEM image from a3RBC-S LC thin film quenched at SmA temperaturerange (87�C in this study), showing domains (num-bered in Fig. 3a for convenience) separated by abruptboundaries. The domains, ranging from a few hun-dreds of nanometers to microns in size, are believed toconsist of smectic layers in different orientations,while the domain boundaries are corresponding to thediscontinuity of the layers. The contrast of the domainsmay come from the differences in layer orientation andthickness. The domains (I, III, and V in Fig. 3a) withvisible layered structure are those at edge-on orienta-tion. The layers inside each domain are basically con-tinuous, but often bent (Fig. 3a). Though the curvatureof the layers is gradual, the orientation change canamount to a large degree with distance. Despite of thecurvature, the layer spacing is very well defined, rang-ing between 3.75 nm and 3.85 nm, as measured fromthe images and the fast Fourier transform (FFT) pat-terns (the inset of Fig. 3a). The result matches the�3.7 nm spacing determined by synchrotron SAXS(Zhang et al., 2012). Figure 3b is a magnified image ofthe marked region in Figure 3a, showing the smecticlayers and the slight bend. A molecular model of thelayered structure is given in the inset of Figure 3b.The shape of the domains can be irregular. For exam-ple, judged by the layer continuity and orientation,domain I extends to the lower right region in Figure 3aand is overlapped with domain III in the thicknessdirection. Figure 3c shows a magnified image of theoverlapping area (I1III), and the corresponding FFTpattern (the inset).

    In contrast to 3RBC-S, 3RBC-N does not have a mac-roscopic smectic mesophase, but a nematic phasebetween 62.5�C and 100.1�C. However, recently therehas been speculation of the possible existence of short-range smectic-C clusters which may likely be the causeof some unusual macroscopic behavior in bent-corenematics (Hong et al., 2010). By quenching nematic3RBC-N thin films at different temperatures duringthe cooling process from isotropic phase, we reportedthe observation of nanometer-sized smectic domains,which was enhanced at lower temperature (Zhanget al., 2012). Figure 4a shows a typical cryo-TEMimage and the corresponding FFT pattern from the3RBC-N thin films, indicating no long range 1D orderbut layered smectic nanoclusters in the nematic sub-strate. The smectic clusters are normally a few tens ofnanometer in length and consist of 1 to 10 layers. Thelayer spacing of the clusters is not rigid and mostlyvaries between 2.8 nm and 3.1 nm, which is independ-ent of the quenching temperature. The considerablysmaller spacing when compared to 3RBC-S can be con-sidered as an evidence for a tilted director, i.e., theformation of a SmC structure, as proposed by Honget al. (2010). Similar to the abovementioned result for3RBC-S, the layers can also bend, especially in largerclusters, and overlapping of clusters in the thicknessdirection can be observed (as indicated by solid arrowsin Fig. 4a). The existence of smectic nanoclusters inthe nematic phase, including well-defined single

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  • layers, indicates clearly the strong tendency of bent-core LC molecules to close pack into layers. Thoughvery different orientations can be found in neighbor-ing clusters due to the local director fluctuation(Zhang et al., 2012), notable orientation preferencecan be identified frequently in local areas, probablyowing to the consistent director alignment as thesmectic clusters evolve from the nematic substrate.Figure 4b shows a local region with the majority ofthe clusters having similar orientation. Figure 4cshows that some clusters with almost the same orien-tation are assembled into a large cluster with morethan 40 layers. Such large assemblies can only befound rarely, but small scale side-by-side assemblies

    often with very similar orientations seem to be a com-mon characteristics of the smectic nanoclusters. InFigure 4a, we use hollow arrows to point out a few ofthem. The lower insets of Figure 4a are magnifiedimages of two assemblies enclosed by dashed squares.An edge dislocation-like extra half layer can be identi-fied in the left inset. While in the right inset, a cleargap of a few nanometers in width can be seen in thelower part of the assembly where the layers of the twosides are not aligned along the layer normal, i.e., outof phase.

    Besides the simple cases shown above, complicatedsituations also present frequently, leading to morecomplex assembly structures. For example, some

    Fig. 3. (a) A cryo-TEM image revealing domain structure in a3RBC-S thin film quenched at 87�C. The inset is the correspondingFFT pattern. The domains are numbered for convenience. (b) A mag-nified image of the local region identified by a dashed square indomain I of Figure 3a. The inset shows a schematic of the moleculearrangement in the proposed SmA layer structure, and the arrow

    indicates the layer normal. Note that the molecules can rotate freelyaround the long axis at the absence of external fields. (c) A magnifiedimage and its corresponding FFT pattern of the overlapped area ofdomains I and III (Fig. 3a). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

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  • neighboring clusters have different layer numbers ordifferent orientations, and some assemblies involvemore than two clusters. Figures 4d–4g show a few rep-resentative complex assemblies. By using the snapshotspecimens, it is difficult to determine the lifetime ofthe smectic clusters or the sequence and dynamics ofthe processes. However, the detailed structurerevealed by direct TEM imaging, e.g., partially con-nected assemblies and adjustments of the layers nearthe cluster joints, seems to suggest that the moleculesbetween the side-by-side clusters may align them-

    selves in layers and form junctions to connect theneighboring clusters. Such later-stage “bridging” pro-cess may account for a major part of the complexassembly structures in addition to possible growthdefects in individual clusters. Figure 4d shows prob-ably a fully connected assembly consisting of at leastthree small clusters which are roughly “in phase”. Theupper junction (marked by a hollow arrow) is smooth,while the lower one bends sharply because the connec-tions of layers shift by one layer along the layer nor-mal, i.e., the smectic layers in the junction have adifferent orientation from that of the clusters. Theupper junction marked in Figure 4e is between twoparallel clusters shifted by half of the layer spacing,while the lower junction involves three clusters. Notethat the two junctions are only 1–3 nanometers inwidth, even narrower than the layer spacing, and theleft side of the lower junction seems to be incomplete,i.e., the alignment of the molecules in the gap may bestill in progress before the freezing process. In addi-tion, a short extra layer (pointed out by a solid arrow)is formed between the two upper clusters that tilt fromeach other by about 18�. Edge dislocation-like addi-tional layers can be found when misalignments occurbetween the layers in different clusters, which is espe-cially common when the two clusters have differentlayer numbers or orientations. Figure 4f showsanother junction of three clusters. An extra half layeris indicated in cluster I as it forms a sharp bend withcluster III that is tilted by �40� from cluster I. Thoughthere is only �15� tilt between clusters II and III, twoextra half layers can still be identified (Fig. 4f). Figure4g shows a relatively big assembly in which the twoclusters are parallel to each other and only slightlyout-of-phase. Three extra half layers are marked,which are mainly caused by the changing orientationsof the “bridging” layers in the junction. The non-parallel “bridging” layers may be partly caused by thefluctuation of the molecule orientation. The detailedstructures in Figures 4e–4g also suggest that some ofthe layers are bridged to two of the layers in the neigh-boring cluster instead of the normal “one-on-one” cor-respondence. In addition, the local layer spacing nearthe complex junctions can largely deviate from theaverage value (�2.95 nm). For example, we haveobserved layer spacings smaller than 2.5 nm or largerthan 4 nm. The successful observation of the abovedetailed structures demonstrates the unique advant-age of direct TEM imaging combining high resolutionand projection along the whole thickness of a thinspecimen.

    In Figures 5a–5e and 6a, we show cryo-TEM tex-tures of the lyotropic chromonic LCs (DSCG and SSY)prepared by the technique of thin film plunge freezing(Fig. 2d). At the quenching temperature (room temper-ature), the chromonic LC samples exhibit isotropic(6.2% DSCG) and uniaxial nematic (15% DSCG and30% SSY) phases, respectively, revealed by PLM (notshown). The uniform contrast of the suspended 6.2%DSCG thin film (Fig. 5a) and the stripes with bright/dark contrast in 15% DSCG (Fig. 5b) match the iso-tropic and uniaxial nematic structures, respectively.The dark stripes can be understood as the elongatedchromonic aggregates formed by face-to-face packingof the DSCG molecules in water. Figure 5c shows the

    Fig. 4. (a) A typical cryo-TEM image showing the smectic nanoclus-ters in 3RBC-N nematic thin films. The narrow solid arrows point outa few overlapping clusters, while the hollow arrows denote assembliesof side-by-side clusters. The upper inset is the corresponding FFT pat-tern. The lower insets are the magnified views of the 30 nm 3 30 nmjunction areas marked by squares. An extra half layer is pointed outby a solid arrow. (b) A representative region of smectic clusters withpreferred orientation. (c) A rare local area with a large smectic assem-bly over 40 layers. (d–g) Magnified images of representative complexassemblies. The junctions are marked by hollow arrows, and the extrahalf layers by solid arrows. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

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  • corresponding FFT pattern of the nematic texture inFigure 5b, while Figure 5d is a magnified image of themarked local area. The length of the columnar aggre-gates was measured to be 25–80 nm. The values matchreasonably well the estimated upper limit of aggregatelength, 20 nm, in the isotropic phase of 14% DSCGimmediately above the nematic-to-isotropic phasetransition (Nastishin et al., 2004). The spacingbetween the stripes varies from 3.6 nm to 5 nm, andaverages around 4.2 nm, which matches the measuredvalue from the FFT pattern. The spacing of the aggre-

    gates measured from the side-views can be underesti-mated due to the projection effect. In addition, theaggregates seem to assemble into some bundles of tensof nanometers in size, which rules out any long rangeorder and may partly account for the diffused brightspots in the FFT pattern. The nematic arrangement ofthe aggregates is also evidenced by the frequent obser-vation of groups of dark dots (Fig. 5e), which should besmall bundles of aggregates oriented perpendicular tothe specimen surface. The relative positions of theaggregates in each bundle are rather random,

    Fig. 5. (a–e) Cryo-TEM results of DSCG lyotropic chromonic LCsprepared by the “thin film” approach (plunge frozen specimens). (a) Atypical image of 6.2% DSCG. (b) A typical image of 15% DSCG. (c)Corresponding FFT pattern of the nematic structure shown in Figure5b. (d) A magnified image of the marked local area in Fig. 5a. (e) Animage of the aggregates perpendicular to the thin film surface

    observed in 15% DSCG. (f) and (g) CEMOVIS images of nematicregions in 15% DSCG with 10% dextran. The hollow arrow in (g)points out a domain of aggregates perpendicular to the specimen sur-face. [Color figure can be viewed in the online issue, which is avail-able at wileyonlinelibrary.com.]

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  • consistent with the nematic structure. It is expectedthat in the hexagonal columnar phase, at higher con-centration, the aggregates would form a lattice. Thedistance between neighboring aggregates, measuredfrom such top-views, ranges between 4 nm and 5 nmwith an average around �4.6 nm, slightly larger thanthe measured spacing using the side-view images andmatching the XRD measurements closely (Collingset al., 2010; Tortora et al., 2010). The DSCG samplescontain a relatively small amount of organic materials,only 6.2 wt% and 15 wt%. Because of the high watercontent and low viscosity, the TEM specimen prepara-tion by thin film plunge freezing, can be performed in amanner similar to the preparation of aqueous biologi-cal samples.

    Figure 6a shows a cryo-TEM image of the SSY solu-tion, in which the percentage of the organic componentis higher, about 30 wt%. With the decrease of the watercontent, the lyotropic nematic phase becomes ratherviscous, making it challenging for the blotting processbefore the plunge freezing. As a result, prolonged blot-ting or larger blot force are often required to make athin specimen, which may cause change in water con-centration and modification of the LC structure.Reduced amount of solution (

  • perpendicular bundles can only be observed at theedge of the holes and mostly consist of small numbersof aggregates. While in the bulk approach, differentaggregate orientations can be more equally seen, andthe bundle size is often a few hundreds of nanometersor even larger (Fig. 5g). Besides, the local orientationof an aggregate bundle observed in bulk approachoften varies slightly from the nearby ones, and nearly90� variation has been observed in the SYY sample. Inaddition to the technical challenges involved, a majordisadvantage of the bulk approach is the artifacts (e.g.,knife marks, crevasses, and compression) which can beintroduced during cryo-sectioning (Han et al., 2008;Studer et al., 2008). In contrast, the plunge freezing ofthin films usually results in cleaner TEM specimenthus TEM images without noticeable artifacts. In Fig-ure 2f, FFTEM is also shown as an alternative to theCEMOVIS after the high-pressure freezing process. Itshould be mentioned that our initial attempt ofFFTEM and cryo-SEM (not shown) on the 15% DSCGsample failed to yield columnar aggregate-relatedstructures, probably due to the facts that the fracturesurface did not correlate closely with the aggregatesand/or the replication process did not provide sufficientresolution.

    Considering the overall similarity of the results gen-erated, we believe that thin film and bulk approachescan both work for a wide range of lyotropic LCs if usedproperly. To avoid the complications of cryoprotectants,thin film plunge freezing is preferred for samples withhigh percentage of water. While for samples with lesswater, the bulk CEMOVIS approach using high-pressure freezing and cryo-ultramicrotomy is morefavored to get reliable results. It should be also men-tioned that for very viscous lyotropic LCs, thin filmplunge freezing and “bulk” high-pressure freezing mayboth encounter great technical challenges. An ongoingeffort is being made for such highly viscous lyotropicmaterials.

    Complementary Combination of DirectCryo-TEM Imaging and Indirect FFTEM

    As described earlier, LC materials are probably oneof the most complicated samples for TEM studies.There is basically no one-for-many routine at the cur-rent stage. The available techniques, often developedinitially for bio-materials, all have obvious advantagesand disadvantages when applied to LCs, as summar-ized briefly later in the paper. On the other hand, thedifferent techniques are complementary, and theircombination can provide comprehensive characteriza-tion for many LCs. In this study, we performed cryo-TEM using both thin film and cryo-sectioning, andFFTEM on thermotropic CB7CB and lyotropic DSCG.Here we present results from complementary FFTEMand cryo-TEM revealing the nanoscale structure of thepeculiar nematic phase, the so-called twist-bendnematic (Ntb) in CB7CB with molecules of a dimertype.

    As Borshch et al. (2013) summarized, there can bethree types of nematic structures. Besides uniaxialnematics (N) introduced earlier, another well-knownmesophase is the chiral (cholesteric) nematic phase(N*) in which the director forms a right-angle helix,

    twisting in space around the helical axis and remain-ing perpendicular to this axis. While in the strikinglydifferent structure Ntb as predicted by Meyer (1976),Dozov (2001), and others (Memmer, 2002; Shamidet al., 2013), the director forms an oblique helicoid,being tilted (rather than perpendicular) to the axis oftwist. Figure 7a shows schematics of dimeric moleculesforming a single spiral (left-handed) and filling a smallspace in the proposed Ntb structure. Note that in bothN* and Ntb, director twist does not imply modulateddensity of the material (in contrast to the case of smec-tics, in which the layered structure is associated withthe density modulation). Thus, the 1D periodic struc-ture along the helix axis is defined only as a periodicchange in local molecular orientation not associatedwith any detectable modulation in mass or electrondensity (Borshch et al., 2013; Chen et al., 2013; Cestariet al., 2011; Panov et al., 2010). The most striking fea-ture of Ntb helical structure is that its period is verysmall, on the order of 10 nm, which is by two orders ofmagnitude smaller than the pitch of the chiral nem-atics formed in materials such as cholesterol deriva-tives. As a result, the nanoscale periodicity of the Ntbphase has been established only very recently, in 2013,thanks to TEM experiments (Borshch et al., 2013;Chen et al., 2013). Moreover, the TEM experimentsallowed Borshch et al. (2013) to establish that thestructural organization follows the geometry of theoblique helicoid rather than, for example, a mixedsplay-bend state, also predicted theoretically (Dozov,2001; Memmer, 2002; Meyer, 1976; Shamid et al.,2013).

    To demonstrate such a 1D periodic structure, thesurface sensitive FFTEM turned out to be essentiallyuseful. Though the low-temperature fracture can occuralong various orientations, it is easier to fracture alongthe length of the molecules since less energy/area isrequired to break side-side bonds (Berreman et al.,1986). In CB7CB, the favored fracture planes are thoseparallel to the helix axis, so it is possible to visualizethe 1D periodic structure if smooth fracture and propershadowing can both be achieved. In practice, suchdesired fracture and replication do present and well-defined periodic layers can be observed (Fig. 7b). Thelayer spacing is considered to be corresponding to thepitch of the helix structure (Borshch et al., 2013; Chenet al., 2013). Figure 7b shows clearly domains of sev-eral hundreds of nanometers to microns in size. Mostlayers are either very straight or slightly curved (theinset of Fig. 7b). Several expected fracture configura-tions have been observed. If the fracture occurs almostperfectly parallel to the helix axis (parallel fracture),clean layer structure can be revealed and the pitch canbe measured accurately (domain I in Figs. 7b and 7c).If the fracture surface has a tilting angle relative tothe helix axis (oblique fracture), steps can be formedwith the terraces still showing basically the same fea-tures as the parallel fracture (domain II in Fig. 7b).

    As an alternative to the steps in oblique fracture,Bouligand arches (Bouligand et al., 1968) can beformed for helix structures such as the Ntb phase, asshown in Figures 7d and 7e corresponding to differentfracture angles. The arch structure observed inCB7CB is very different from the arch structureobserved in chiral nematics, as discussed in details by

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  • Borshch et al., (2013). In the chiral nematic phase,each 180� twist of the director results in a completearch, in which the director imprint rotates (throughin-plane bend and splay) by 180�. In contrast, theoblique helicoidal structure of the Ntb phase results intwo other types of Bouligand arches: (i) an alternatingsystem of “wide” and “narrow” arches (Fig. 7d), each ofwhich corresponds to a 180� twist, and (ii) a periodicsystem of incomplete arches, in which the imprint doesnot reorient by 180� (Fig. 7e). Neither of the two lattertypes of the Bouligand arches is characteristic of a chi-ral nematic. Note that the wide and narrow arches inFigure 7d have a different affinity to fracturing andsubsequent metal deposits, thus they might show a dif-ferent contrast in the resulting FFTEM textures ofreplicas.

    The above results indicate clearly a 1D periodicstructure at the fracture surface and a helix axis in thebulk material. As Chen et al. (2013) reported, the layercontrast in CB7CB is considerably weaker compared toother layered LC systems with mass and electrondensity modulation. Similar absence of density modu-lation was reported by Borshch et al. (2013) for

    another Ntb material. For parallel fracture, the periodin most of the domains ranges between 8.0 and 8.2 nm.In Figure 7b, the pitch was measured to be 8.05 nmfrom the image and the FFT patterns. In addition tothe domains with narrow pitch distribution, largerpitches can also be found (e.g., 8.6 nm), which can bepartly attributed to the cosine relation between thelayer spacing in a parallel fracture and the measuredspacing in oblique fracture. An interesting finding isthat some domains show smaller pitches especially inthe peripheral regions, in contrast to the uniform pitchfound in most of the individual domains (e.g., Fig. 7b).Figures 7f and 7g show two such domains, where thepitches near the center of the domains are �7.6 nm,only slightly smaller than the normal ones, but themeasured periods near the boundaries are

  • Ntb phase, which may adopt different conformationalstates with small energy difference depending on thelocal environment. It is also worth mentioning thatsome detailed features of �1 nm in size and distanceare resolved near the boundaries, though ratherpoorly, suggesting the potential use of FFTEM in highresolution imaging in some special LC systems.

    The free surface of CB7CB sets the director orienta-tion perpendicular to itself (the so-called homeotropicalignment), which makes suspended thin films suita-ble to study the top-view structure. Figure 8a is a typi-cal cryo-TEM image of suspended CB7CB thin filmsquenched at 95�C, showing clearly column-like struc-ture and narrow low-density areas between some ofthe columns. The columns tend to have more or lesscircular shape and have an average size of �27 nmmeasured over a large area. Our results also show thatthe column structure from the suspended films can beinfluenced by thermal treatment and the film thick-ness. Careful examination of the FFTEM imagesreveals the side-view of the column-like texture insome local regions. For example, in Figure 7b, we usearrows to denote the column boundaries from the topof the layered domain, emphasizing the distinct col-umn shape near the domain boundaries. The columnboundaries can be seen as narrow areas often with adiscontinuity of the layered structure and elongatingroughly along the layer normal in the image, which isalso revealed by the diffuse scattering perpendicular tothe ordering direction in the FFT pattern. In theimage, the 8 marked adjacent column boundaries havean average distance of �26 nm. We have carefullyexamined the layer structure across the column boun-daries in the FFTEM images. Along with a few excep-tions, most of the layers exhibit no shift along thelayer normal, which may mean that the helix structuretends to stay “in phase” for neighboring columns. Theinset of Figure 7c is a low-pass filtered gradient imageof the marked rectangular area, which enhances thelayer characteristics and confirms the “in phase” con-figuration. The top-view of the columns can also beidentified in the FFTEM images. The inset in Figure8a is a typical local region with a cluster of circular col-umns, corresponding to a fracture perpendicular to thehelix axis. It should be pointed out that it is difficult tocorrelate such features in FFTEM to columns without

    the cryo-TEM results. Except those layer-related fea-tures, the FFTEM images tend to be complicated atthe presence of artifacts and various features relatedto the fracture morphology.

    Despite of the consistency discussed above, the ori-gin of the column structure cannot be determinedunambiguously at this stage. The columns do notappear to be as common in the FFTEM images as inthe thin films if we assume that the subtle feature canbe well replicated in the specimen preparation process.In addition, such “leopard” pattern can sometimes befound in plunge-frozen cryo-TEM specimens withwater-based buffer due to the local concentration vari-ation (e.g., loss of water). Though the same cause doesnot apply to the single-component CB7CB, the columnstructure may still be an unusual feature in the bulkmaterial but enhanced greatly in thin films.

    Our current results suggest that the column struc-ture with their long direction along the helix axis maybe introduced by the coexistence of right- and left-handed Ntb columns, while very small amount of resid-ual high temperature N phase may exist at the columnboundaries (Borshch et al., 2013). A strong evidence isthat upon doping with chiral dopant (1–1.5% right-handed limonene), the column size increases dramati-cally (Fig. 8b) under the same thermal processing con-dition, as the chiral dopant keeps more columns in thesame handedness. The columns in the doped filmsoften have irregular shapes. In Figure 8b, a columnwith typical shape is highlighted, which shows thatthe smallest dimension of the columns is very similarto that found in the pure films, but a few nearby col-umns may merge together due to the same chirality. Tocompare the column size, we simply compare thedomain numbers in regions with the same area forboth pure and doped films. For example, for a 200 nm3 200 nm area, 50–60 columns can be found in purefilms, while only �20 or even less in doped films.Recently, Hoffmann et al. (2014), proposed a similarmodel based on nuclear magnetic resonance (NMR)measurement of a similar LC compound, wherein themolecules organize into highly correlated microscopicdomains of opposite chirality.

    Another possibility of the formation of the columnstructure observed in thin films is due to the confine-ment of the thin films. As the recent simulation(Fukuda and �Zumer, 2011) showed, two-dimensionalSkyrmion lattice embedded in topographic defects candevelop in a highly chiral nematic LC thin film withsurfaces favoring homeotropic alignment. The two pos-sibilities discussed above both indicate a high chiralitystructure, consistent with the proposed twist-bendnematic structure.

    In addition, our initial cryo-TEM result (not shown)using “bulk” cryo-sectioning routine (Fig. 2b) did notreveal any layered structure consistent with theFFTEM result. This can be considered as a further evi-dence that there are no modulation of electron/massdensity for the Ntb phase. The above comprehensivestudy combining FFTEM and cryo-TEM clearly showsthat the low-temperature nematic phase of CB7CB has1D periodic helix structure and match the predictedNtb. The successful revealing of the 1D periodic struc-ture is attributed directly to the replication of thesmoothly fractured surface in FFTEM.

    Fig. 8. (a) and (b) Typical cryo-TEM images from suspended thinfilms of pure CB7CB and CB7CB doped with right-handed limonenerespectively. The inset in (a) shows a typical FFTEM image of a clus-ter of column-like circular features, which may be the top view of thecolumns in FFTEM as the fracture is perpendicular to the helix axis.[Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

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  • Surface/Thickness Effect, Cooling Rate, andRadiation Damage

    The routines summarized in Figure 2, includingcryo-TEM and FFTEM, all aim at taking snapshots ofmicrostructures of LC materials. To be successful, sev-eral critical requirements have to be satisfied for both“thin film” and “bulk” approaches: (1) the pre-freezingsamples should have the desired native structure; (2)the samples have to be quenched effectively at a suffi-cient cooling rate to prevent from any further phasetransition and structure change during and after thefreezing process; (3) any possible misleading structuredeformation or change introduced during the specimenprocessing after the quenching should be kept mini-mum. For thin film approach, the main concern is tomake thin films with representative structure. Thelimited thickness may lead to undesired surface/inter-face effect. While for the “bulk” approach, it is oftenchallenging to achieve sufficient cooling rate to quenchthe structure effectively. Damage and deformation canalso be introduced during the processing of the “bulk”material into TEM specimens. In addition, direct TEMimaging routines all suffer from radiation damage,which may become a serious issue for those highly sen-sitive materials.

    As already indicated, the surface that LC makescontact with can often strongly influence the molecularorientation near LC surface, i.e., the so-called surfaceanchoring, which can be simply understood as epitax-ial growth of solids on substrates (J�erôme, 1991).Depending on the substrate surface configuration, theLC molecules can be perpendicular (homeotropic), par-allel (planar) or tilted relative to the substrate surface.If the LC film is too thin, the internal structure can bedominantly governed by the surface and does not rep-resent the bulk structure which is often desired inTEM studies. Obviously, the surface effect has pro-found influences on the TEM results using thin filmapproach and deserve more detailed studies. Here wepresent some of our results to demonstrate the effect.

    For the thin film approach used here, there exist twoLC/air interfaces for a suspended LC film (if ignoringthe influence of the hole edges in the supporting car-bon film), while one LC/air interface and one LC/car-bon interface for supported thin film. Simpletreatment of the carbon film can affect the LC struc-ture greatly. Figures 9a and 9b compare the CB7CBthin film deposited on plasma treated and untreatedthin carbon grids. The plasma-treated carbon exhibitsmuch better wetting property to the CB7CB, resultingin large area of relatively uniform thin film. Similar tothe suspended films (Fig. 8a), the CB7CB forms a col-umn structure in the whole film (Fig. 9a). The upperinset of Figure 9a shows a magnified image of a localarea, showing clearly sharp bright lines in the elon-gated columns and bright dots in relatively circularcolumns, respectively. The lines are roughly along theorientation of the long direction of the domains. Suchbright lines/dots are not observed in suspended films(Fig. 8), and are considered as some low-density topo-graphic defects formed due to the different moleculealignments at the two surfaces. The lower inset of Fig-ure 9a is a plausible model showing the side-view of atoroidal focal conic domain. The arrows represent the

    helicoid axes with the same handedness in the domain,which are tangential to the CB7CB/carbon interface,but perpendicular to the free surface. Note that the hel-ical axes are more or less straight due to the periodictwist-bend structure. AA is a defect line in an axially-symmetric circular column, and a narrow defect wall(with its long direction along the projection direction) inan elongated column, corresponding to the bright dotsand lines in Figure 9a, respectively. On the other hand,on the untreated film, the LC material typically tendsto form featureless droplets with increasing thickness

    Fig. 9. (a) A cryo-TEM image of a CB7CB thin layer supported byplasma-treated continuous carbon film. The upper inset image mag-nifies the marked local area, showing the sharp dots and lines insidethe columns. The lower inset is a schematic model of the formation ofthe sharp dots and lines. (b) A cryo-TEM image of a CB7CB dropleton untreated continuous carbon film. (c) A cryo-TEM image of 3RBC-N thin film with a gradual increase in thickness from the film edge tothe inside. The inset image magnifies the marked thick area (>500 nm in thickness). (d) Intensity profile along the line drawn inFigure 9c, showing the variation of the thickness. The inset is anintensity profile along the line in the inset of Fig. 9c, demonstratingthe modulated intensity of the layered cluster along the layer normal.(e) A cryo-TEM image of a thin part of the 3RBC-N thin film withvery narrow smectic clusters. (f) A cryo-TEM image of a plunge-frozen DSCG thin film. The holes in this area are relatively small.The lacey carbon frame and several bundles of columnar aggregatesperpendicular to the liquid surface are marked. The area marked bya dashed square is corresponding to the area in Fig. 5e. [Color figurecan be viewed in the online issue, which is available at wileyonlineli-brary.com.]

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  • from the edge to the center. The columns of �20–40 nmin dimension can only be seen in thick droplets andbecome a less pronounced feature as the thicknessincreases further, as seen in Figure 9b. Bright lines/dots can still be identified at the column centers. Theabove result may indicate that the carbon film (treatedor untreated) favors director orientations other thanthe homeotropic configuration for CB7CB, so the influ-ence of the interface with air can only be seen when thematerial is far enough from the carbon film. On theother hand, the current result may also suggest thatthe column structure is only stable in certain thicknessrange, which should be able to be clarified with morequantitative structure-thickness correlations.

    The strong surface effect in LCs raises doubts on thegeneral use of the thin film approach and make it oftennecessary to discuss the validity of the observedresults. In practice, this can be done by comparingresults in films with different thicknesses by deposit-ing different amount of LC solution on the carbonfilms. Even more conveniently, wedge-shaped areaswith gradually changing thickness can be found fre-quently. For example, Figure 9c shows an image of the3RBC-N thin film with smectic clusters. The intensityprofile can be considered as a rough indication of thevarying thickness, which can be further correlated tothickness through EELS or thickness mapping usingenergy filtered TEM. No smectic layers can be found inthe very thin region, ruling out the possibility that theformation of the smectic clusters are due to surfaceeffect (Ruths et al., 1996). Instead, the surface effect atleast prohibits the formation of the smectic clusterswith layers perpendicular to the surface. It can also beseen that the layered clusters can still be resolved inthe very thick part of the sample (the inset of Fig. 9c).EELS measurement showed that the thick part shouldbe at least 4 times of the inelastic mean free path, i.e.,thicker than 500 nm. The layer length and layer num-ber in the thick area are basically the same as thosefound in thinner regions (100–200 nm). Clusters withvery few layers (e.g., 2) can also be seen in the thickparts of the specimen. Above results show that theobserved smectic clusters stay the same for a widerange of thickness, which validates the application ofthe thin film approach in this material.

    Interestingly, in some of the thin areas, very narrowsmectic clusters can be found (Fig. 9e). The layer widthcan be even shorter than 3 nm, while the layer numberwithin the clusters varies from 1 to 9, which is compa-rable to those in the normal clusters. The layer spacingis considerably larger (3.1 nm–3.5 nm) compared tothe wider clusters. Because the confinement is in thethickness direction, it may be reasonable to assume asimilar cluster depth in the thickness direction. Con-sidering that the interface prevents the layered struc-ture formation, the short layers smectic clusters mayindicate the rather strong tendency of the 3RBC-Nmolecules to form layered structure as the surfaceimpact becomes weaker.

    Carbon films also play an important role in theobserved structure of suspended chromonic lyotropicLC thin films prepared by plunge freezing (Figs. 5a–5eand 6a). As we mentioned earlier, most columnaraggregates tend to orient along the long direction ofthe holes, and probably prefer to be parallel to the

    liquid surface. Inside each hole, the aggregates havebasically the same orientation except that the shape ofthe hole affects those aggregates near the edge. Thiscan be seen more clearly in the region with smallerholes, as shown in Figure 9f. The areas near hole edgesare under the competing influences of the liquid sur-face and the carbon edge. As a result other than lyingalong the edge and parallel to the liquid surface, smallaggregate bundles perpendicular to the liquid surfacecan be found near the hole edge (Fig. 9f).

    In this study, we only employ LC surfaces makingcontact with air and carbon films. In principle, a vari-ety of materials and surface treatments can be used.In addition, the surface/interface impact may providea way to modify the LC structure or orientations usingthe thin film approach or simulate the working condi-tion of LC devices in TEM studies.

    In general, it is more difficult to quench sampleswith their internal structure intact for bulk samplesthan thin films since the cooling rate inside the sampleis mainly determined by the heat conductivity of thematerial, and more difficult for lyotropic LCs thanthermotropic LCs due to the high percentage of waterwhich has unusually high heat capacity. For example,to vitrify a water based sample requires a cooling rateof the order of 105 K/s. In practice, it is straightforwardto use liquid ethane as cryogen for both thermotropicand lyotropic LC thin films. Thermotropic LC thinfilms normally require a less demanding cooling ratedue to considerably slower molecular diffusion andsmaller heat capacity. As a result, the much more con-venient quenching in liquid nitrogen often gives thesame results as liquid ethane, revealed by comparingthe results using liquid nitrogen and liquid ethane ascryogens (not shown).

    Generally speaking, plunge freezing of bulk materi-als may only work for samples of a few microns inthickness. However, thinner samples may experienceincreased surface effects and also make it challengingfor the follow-on processing (e.g., cryo-sectioning andfreezing fracture). The application of high-pressurefreezing initiated in this study seems to be a reliablemethod for bulk lyotropic LCs up to a few hundreds ofmicrons in the smallest dimension. More efforts arestill needed to improve the structure preservation inhigh water percentage samples, clarify the influence ofthe cryoprotectant, and reduce the damages in the fol-lowing cryo-sectioning process. For thermotropic LCs,currently there is no equivalent technique to high-pressure freezing for lyotropic LCs to allow structurequenching at a lower cooling rate. Fortunately, forsome of the thermotropic LC materials, e.g., CB7CB(Chen et al., 2013), transitions can be supercooled,which helps to preserve the native structure even at aslow cooling rate. But for other materials withoutsupercooling, extra caution has to be taken. In prac-tice, different structure indicating different coolingrate can be observed at different depth using the com-bination of plunge freezing and cryo-ultramicrotomy(not shown).

    Direct cryo-TEM imaging techniques used here allsuffer from radiation damage to the electron-transparent specimens. Our results show that the radia-tion damage of the LC thin films first exhibits as the dis-sipation of the ordered structure (smectic layers in this

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  • study) due to the very weak intermolecular force stabi-lizing the ordering. Such subtle damage can often leadto the overlooking of the ordered structures present, asit does not involve observable structure and shapechanges at a large scale. In some materials, the damageto the ordering may happen at very low doses (even< 2e2/Å2), making it difficult to capture an image with rea-sonable S/N ratio. On the other hand, those mass lossrelated damages, leading to larger scale structurechanges, occur at much higher dose. As a result, atomicresolution imaging can often be obtained before anyobvious shape changes, provided ordered structures arenot involved or not the target of the investigation. Forexample, Figure 10a shows a lattice image of gold nano-particles doped in 5CB. The 5CB thin film also exhibitssufficient stability for spot-mode STEM imaging andspectroscopy (e.g., EELS and EDS) measurement. Onebenefit of STEM is to increase the visibility of some fea-tures in thick regions owing to the emphasis of atomicnumber difference and the minimized influence of crys-tal orientation on the contrast. Figure 10b shows abright field TEM image of gold nanoparticles dispersed

    in a thick region of 5CB. The small nanoparticles can behardly identified. In contrast, a STEM Z-contrast imageof a similar area (Fig. 10c) shows an abrupt contrast evenfor nanoparticles of �1 nm in diameter. Figure 10dpresents the corresponding EDS spectra taken by scan-ning a nanometer-sized box over the top of an individualnanoparticle (Gao et al., 2003b) and the surrounding5CB. As organic materials, the radiation damage of LCmaterials basically fits into the description given byEgerton et al. (2004). Several ways can be considered tominimize the damage: lowering the dose combined with amore sensitive camera, use of a higher beam energy andof a thicker specimen. In practice, field emission electronsource often causes more irradiation damage. A tradi-tional LaB6 source can reduce the damage yet still keep asufficient resolution for LC studies (Egerton et al., 2004).

    A Brief Comparison of the Different SpecimenPreparation Techniques for LCs

    In this study, we aim to establish an easily accessibletoolset for the TEM specimen preparation of a wide

    Fig. 10. (a) A TEM image showing the lattice of Au nanoparticlesembedded in 5CB. The marked spacings are corresponding to Au (111)and (200) planes which have spacings of 2.36 Å and 2.04 Å in bulk Au,respectively. (b) A bright-field TEM image of Au nanoparticles in athick area of a 5CB thin film. (c) A STEM Z-contrast image of Au nano-particles (AuNP) in a thick area of 5CB film, demonstrating greatly

    enhanced contrast compared to the bright-field image. (d) EDS spectraof an individual Au nanoparticle and the nearby 5CB substrate takenby scanning the focused electron beam in a nanometer-sized box toreduce the beam damage and correct the specimen drift. [Color figurecan be viewed in the online issue, which is available at wileyonlineli-brary.com.]

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  • range of LCs. Based on the earlier discussion, a briefcomparison of the different techniques is given inTable 1. At the current stage, there is simply no one-for-many routine. Each method can be powerful forcertain materials, but can also be difficult to apply toothers. For example, direct cryo-TEM allows high reso-lution imaging and the application of a variety of TEMtechniques, but suffers from radiation damage; whilethe replica FFTEM provides basically the oppositeadvantages and disadvantages. In addition to the rou-tines mentioned above, several other techniques, oftenoriginally developed for biological samples, can also beapplied to LC materials. For example, thermotropicthin films can also be obtained by spin coating or sus-pension on water (Voigt-Martin and Durst, 1987),which may result in uniform thickness. Jet freezing(Severs, 2007) and slam freezing (Mondain-Monval,2005) can be used as alternatives in freezing processand may provide more or less higher cooling rate forsome special LCs than plunge freezing. Slam freezingwith liquid helium cooled cooper block has been shownto provide better structure preservation in FFTEM ofsome LC materials (Leforestier et al., 1996). A self-pressurized rapid freezing was proposed using onlyplunge freezing and sealed capillary tubes (Leunissenand Yi, 2009), which may be able to be applied to somelyotropic LCs as a low-cost version of combined highpressure freezing and cryo-sectioning (Fig. 2e).Another interesting specimen preparation techniqueuses similar procedure to the plunge freezing FFTEM(Fig. 2f): a holey carbon coated TEM grid is placedbetween the two planchettes during the freezing pro-cess and some thin areas may remain on the film uponfracture, which can be used for direct cryo-TEM obser-vation (Belkoura et al., 2004).

    The very convenient-to-use thin film approachallows the highest cooling rate and avoids further spec-imen processing after the freezing, which usuallyresults in the best imaging quality and preservation ofthe pre-freezing structure. But surface effect has to beconsidered especially for structures with dimensioncomparable to the thickness. Another practical advant-age of thin film approach is that very small amount ofmaterial is required compared to bulk approach, whichis often important for some thermotropic LCs due topossible complicated synthesis process.

    FFTEM and cryo-sectioning, both using bulkapproach to minimize surface effect, require effectivequenching of thick samples, and often show similarresults (not shown) especially for thermotropic LCsdue to similar freezing process. As a widely used tech-nique, FFTEM effectively avoids radiation damage

    and uses convenient room temperature observation,though the resolution is limited to a few nanometers.We also demonstrate the critical role of the surfacesensitivity of FFTEM in this study, which may makeFFTEM an irreplaceable technique for LCs at the cur-rent stage. Artifacts can be common in FFTEM imagesand extra caution needs to be taken to explain theresults because the replica shadowing/contrast totallydepends on the fracture surface morphology and depo-sition direction, and does not necessarily correlateswith the intrinsic structure in LC materials. The freez-ing of thick sample raises concerns of insufficient cool-ing rate to preserve the intrinsic structures except forthose thermotropic LCs with supercooling property.Plunge-freezing of thick lyotropic LCs leads to watercrystallization, and modification of local structures.(Mondain-Monval, 2005) But FFTEM of lyotropic LCscan still work for low-resolution imaging which is thefocus of most FFTEM studies.

    Cryo-ultramicrotomy seems to combine the advan-tages of both thin film technique and FFTEM, but someof the disadvantages as well. Compared to FFTEM,cryo-sectioning allows high-resolution direct imaging,and the process-induced artifacts (e.g., knife marks, cre-vasses, and compression) are relatively easier to iden-tify. The CEMOVIS technique, combining high-pressure freezing, cryo-sectioning and cryo-TEM,allows the observation of lyotropic LCs beyond the reso-lution of FFTEM. In contrast to the little control of frac-ture process in FFTEM, cryo-sectioning makes itpossible to look at different depth of the sample. As analternative to cryo-sectioning, focused ion beam (FIB)(Strunk et al., 2012) may turn out to be very usefulespecially for some challenging LC materials (e.g., highviscosity lyotropic LCs). The advantages of cryo-FIBinclude less mechanical deformation (knife marks, cre-vasses, and compression), more convenient preparationof specimen in different orientations, and higher effi-ciency in using the regions frozen at fast cooling rate.

    In conclusion, we present our on-going effortstowards high-resolution direct TEM observation ofthermotropic and lyotropic LCs. A series of specimenpreparation techniques have been summarized andtested to provide a comprehensive toolbox for TEMstudies of LC materials. The complications in thesetechniques are also discussed. We demonstrated thatthe current available specimen preparation and low-dose cryo-TEM techniques allow sub-nanometer reso-lution direct TEM imaging of a wide range of LCs,which can become part of the solution to the lack ofeffective structure probe at the molecular level for LCstudies. We also demonstrate that the currently more

    TABLE 1. A brief comparison of different TEM specimen preparation routines for LC materials

    Thermotropic Lyotropic

    Thin film Ultramicrotomy FFTEM Thin film Ultramicrotomy FFTEM

    Freezing technique Plunge Plunge Plunge Plunge High pressure Plunge High pressureDifficulty of specimen preparation Low High Medium Low High Medium HighComplication during freezing Low Medium Medium Low Medium High MediumConsumed material

  • widely used replica TEM technique, FFTEM, is com-plementary to direct cryo-TEM observation. Theircombination is highly recommended to get comprehen-sive information on the LC materials, which can beespecially important at the current stage when thereis no obvious advantageous approach.

    The direct observation of LCs opens ways to apply avariety of TEM techniques beyond low-dose imaging tothese fascinating but challenging materials, for exam-ple, diffraction (Gao et al., 2003a; Zuo et al., 2003,2011), tomography, STEM, and spectroscopy. Maybemore importantly, direct observation beyond takingsnapshots using cryo-TEM may lead to the possibilityof in situ observation of the dynamic processes using insitu TEM. The current development in high sensitivityelectron-counting camera (Li et al., 2013), in situ liquidcell technique (Zhu et al., 2013), and ultrafast TEM(Kim et al., 2008) make it promising to look at LCmaterials with temporal resolution in the near future.

    ACKNOWLEDGMENTS

    The TEM results shown in this article were all takenat the TEM Lab of the Liquid Crystal Institute (LCI)at Kent State University, supported by the OhioResearch Scholars Program Research Cluster onSurfaces in Advanced Materials. MG thanks the LCICharacterization Facility for the support of hisresearch. ODL research is supported by NSF grants(contract grant numbers: DMR-1104850 and DMR-1121288). AK and MGT were supported by the EU pro-ject BIND (contract grant number: 216025). LFacknowledges the National Science Foundation ofChina (contract grant number: 61172011). The authorsthank Dr. Kim Rensing of Leica for the FFTEM andcryo-SEM of high pressure frozen DSCG. MG thanksthe following people for the very helpful discussion:Mr. Michael Boykin from Mager Scientific, Inc., Prof.James Gleeson, Prof. Samuel Sprunt, Dr. Yannian Li,Dr. Chenming Xue, and Mr. Andrew Konya from KentState University; Prof. Haixin Sui from WadsworthCenter, and Dr. Xing Zhang from University of Califor-nia at Los Angeles. Part of the CEMOVIS effort onDSCG was sponsored by Mager Scientific Inc., and per-formed with equipment supported by the MicroscopyImaging Center (MIC) of the University of Bern.

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