High-precision thickness control of ice layer on CVD grown bilayer graphene for cryo-TEM

Carbon 160 (2020) 107e112

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High-precision thickness control of ice layer on CVD grown bilayer graphene for cryo-TEM Ryuichi Kato a, *, Yuri Hatano b, Naoki Kasahata b, Chikara Sato b, Kazu Suenaga a, Masataka Hasegawa a, ** a

Nanomaterials Research Institute, National Institute of Advance Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan Biomedical Research Institute, National Institute of Advance Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 October 2019 Received in revised form 28 November 2019 Accepted 2 January 2020 Available online 7 January 2020

A novel grid for cryogenic transmission electron microscopy (cryo-TEM) was developed using bilayer graphene to facilitate the formation of a thin ice layer over grid holes. To prepare samples, the bilayer graphene adhered on a holey carbon grid was treated by UV/ozone from the top, making the upper graphene layer hydrophilic, while maintaining the high conductivity via the intact lower layer. Virus solution was loaded on the hydrophilized-graphene surface of the grid, and a thin layer was successfully formed and frozen using an automated blotting system. Inspection in a monochromated cryo-TEM at low-acceleration voltage of 60 kV, provided image of viruses embedded in a very thin vitreous ice layer on bilayer graphene surface, at a high signal-to-noise ratio without charging drifts. The vitreous ice layer formed on the hydrophilized-bilayer graphene surface was homogeneously thin and reached as thin as 25 nm, as confirmed using scanning TEM with electron energy loss spectroscopy. Overall, the high hydrophilicity and homogeneous surface free energy of the UV/ozone-treated bilayer graphene is demonstrated to be highly suitable for thin ice layer preparation, and imaging using cryo-TEM, and especially for modern high-performance low-acceleration voltage TEM, which has higher contrast, but demand thinner ice layers on the grid. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction Recent advances in detector cameras and automated cryogenic transmission electron microscopes (cryo-TEM), image processing algorithms including 3D reconstruction softwares [1,2], and innovative cryo-sample preparation method [3] have opened new paths for single-particle reconstruction analysis using cryo-TEM [4,5]. Numerous protein structures that could not have been assumed several years ago are now being solved at high resolution [6]. In the case of single-particle reconstruction analysis using cryo-TEM, it is possible to determine the biological structure without crystallization for X-ray crystallography [7,8] or large amount of purified proteins for an unclear magnetic resonance method. However, the difficulty in preparing the very thin vitreous ice layer with high

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (R. Kato), [email protected]. jp (M. Hasegawa). https://doi.org/10.1016/j.carbon.2020.01.010 0008-6223/© 2020 Elsevier Ltd. All rights reserved.

reproducibility remains as a bottleneck of the cryo-TEM. One of the reasons of the difficulty is attributable to the instability of a buffer layer of less than 100 nm thickness stretched over a carbon hole 1e2 mm in diameter. To avoid the instability of the thin water layer alone, relatively thin hydrophilic amorphous carbon film adhered on a metal grid has sometimes been used instead of simple holly carbon grid for cryo-TEM sample preparation [9], but it generates a high background signal that hindered reconstruction at high resolution. It is also known that the amorphous carbon is semiconductor or insulator rather than metal, hence charge-up of the film at low temperature may lead to distortion and drift in cryo-TEM [10]. Monolayer graphene instead of the carbon film, has been noted as a breakthrough to these issues, it is typically used as an ultrathin single atomic layer (0.34 nm), which minimizes the background signal, but has high electrical conductively. A higher signal-to-noise ratio imaging has been demonstrated with monolayer graphene than with amorphous carbon film [11e13]. In the case of monolayer graphene, however, a problem arises in that preparing a thin ice

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layer is not easy because of film breakage during hydrophilization process [14]. Thus far, numerous attempts have been made to use graphene oxide (GO) as a film materials, GO is characterized by almost perfect electron transparency and by sufficient hydrophilicity for the preparation of vitreous ice [15,16]. In the case of GO, however, because stacking and overlapping of small GO flakes on the TEM grid result in a nonuniform thickness, the fabrication of a mono or bilayer GO membrane is nearly impossible. Furthermore, GO is usually insulating, which may lead to charging-induced image drift [10]. To solve such problems, a controlled small number of graphene layers are required to be hydrophilic while maintaining sufficient conductivity. The chemical vapor deposition (CVD) synthesis of graphene is currently under rapid development [17,18]. High-quality, largearea, monolayer, and bilayer graphene can be synthesized in a controlled manner on a copper (Cu) foil catalyst [19,20]. Bilayer graphene is much more laterally rigid than monolayer graphene and has been shown to have mechanical properties comparable to those of diamond [21]. The excellent mechanical properties of bilayer graphene are expected to satisfy the strict requirements for cryo-TEM sample preparation. In this paper, we attempted to develop a new TEM grid with the ultimate thinness that combines high hydrophilicity and high electrical conductivity. We also report that bilayer graphene grid is very useful for the preparation of thin ice layer samples for cryoTEM.

2. Experimental 2.1. Sample preparation 2.1.1. Bilayer graphene TEM grid preparation The polycrystalline Cu foil was used as a metal catalyst for bilayer graphene. The foil was placed in a plasma-enhanced graphene CVD chamber equipped with a direct Joule heating system and an inductively coupled plasma source [20]. The foil was heat treated for 20 min at 600
C under a hydrogen atmosphere. Methine gas was introduced as a carbon source, and graphene was subsequently deposited via plasma exposure for 90 s at 980
C. The transfer of bilayer graphene from the Cu foil substrate to the supporting grid was performed as follows. A poly (methyl methacrylate) (PMMA) thin layer was spin-coated onto bilayer graphene. After the Cu foil substrate etched with an aqueous solution of HCl and H2O2, graphene with and attached thin PMMA layer was rinsed with ultrapure water several times and then transferred to a commercial supporting grid (Quantifoil®: R2/2 Holey Carbon film TEM grid, Mo 200 mesh). The grid with the thin PMMA layer was dried and then immersed into acetone and isopropyl alcohol to remove the PMMA coating. An ultraviolet/ozone (UV/ozone) treatment was performed using low-pressure mercury lamps. The UV intensity from the lumps was 5.2 mW/cm2, and UV radiation with an extremely short wavelength of 184.9 nm and a short wavelength of 253.7 nm was used. Initially, oxygen was introduced for 10 min to replace the atmosphere, then UV was applied for an arbitrary time.

2.1.2. Cryo-TEM sample preparation A vitrified sample was prepared with TMVs diluted in buffer (3.3 mM of Tris-meleate, pH of 7.0 at 12
C) to a final concentration 3 mg/mL. The UV/ozone treated bilayer graphene TEM gird placed on a FEI Vitrobot Mark Ⅳ. After that the total volume of 1.7 mL was applied on that grid. Subsequently, the grid was blotted for 7s at 4
C and 95% humidity before dipping into the liquid ethane.

2.2. Characterization of the graphene The crystalline quality of the graphene with or without UV/ ozone treatment was evaluated using Raman spectroscopy. A semiconductor laser wavelength of 638 nm and the laser spot size of 1.0 mm was used. The sheet resistance was evaluated by a noncontact sheet resistance measuring instrument using eddy current method [22]. (EC-80, NAPSON Co. Ltd.) The measurement is 20 mm in diameter, and its average value is measured. Initially, the bilayer graphene on Cu foil was transferred onto a 40 mm  40 mm PET film (thickness 188 mm) via a thermal release sheet. Afterward, the average sheet resistance of the bilayer graphene without and with the UV/ozone treatment was obtained by measuring five times at the same point. The water contact angle was measured using the half-angle method (Dropmaster501, Kyowa Interface Science Co. Ltd.) to evaluate the graphene surface hydrophilization. Ultrapure water was used for droplets. The evaluation was performed using the average value of five samples under the same UV/ozone treatment conditions. 2.3. Microscopy Scanning transmission electron microscopy (STEM) was performed using a JEOL JEM2100F TEM equipped with a DELTA aberration corrector operated at 60 kV The sample was heated to 500
C using in situ heating holder to prevent surface contamination. The vitrified sample was maintained at liquid nitrogen temperature during sample transfer and image acquisition in a transmission electron microscope using a cryo-transfer holder (cryoholder- 626, Gatan, USA) The sample temperature was carefully monitored and maintained at approximately 175
C within the microscope using a Smart Controller (Gatan, USA) during experiments. Cryo-TEM experiments were performed using a JEOL TEM system equipped with a Schottky field-emission gun, delta correctors, and a double Wien filter monochromator (3C2) and operated at an acceleration voltage of 60 kV. The TEM images were recorded with a Gatan OneView CMOS camera, and scanning transmission electron microscopy with electron energy-loss spectroscopy (STEM-EELS) was performed using GIF Quantum imaging system for imaging and spectroscopy. 3. Results and discussion To make the bilayer graphene surface hydrophilic, it was subjected to the UV/ozone treatment. UV lamps with wavelengths of 184.9 and 253.7 nm (energies of 647 and 472 kJ/mol) were used to irradiate the bilayer graphene surface. Graphene is composed of carbon sp2 bonds, consisting of s and p bonds with dissociation energies of 348 and 261 kJ/mol, respectively. By breaking the carbon sp2 bond under UV irradiation, oxygen, and hydrophilic groups such as OH and COOH were introduced, rendering the bilayer graphene hydrophilic [23,24]. Fig. 1(a) shows the typical Raman spectra of graphene on Cu foil, the spectra were measured before and after UV/ozone treatment as an index of crystallinity. The G and 2D bands due to graphene are observed in all of the spectra. The intensity of the G band is approximately three to four times greater than that of the 2D band. Fig. 1(b) shows the detailed analysis of the 2D band. All of the 2D bands are composed of the sum of four Lorentzian peaks, indicating AB-stacked bilayer graphene [25]. A slight D band due to defects was observed after UV/ozone treatment. The intensity of the D band tends to increase with increasing UV/ozone treatment time, indicating that the number of defects increases with increasing UV irradiation time. However, the AB stack structure is almost

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Fig. 1. (a) Typical Raman spectra of bilayer graphene with and without UV/ozone treatment. (b) 2D-band fitting analysis using Lorentzian peaks. (A colour version of this figure can be viewed online.)

maintained. To evaluate the uniformity of the graphene layer number, Raman spectra were taken from graphene on Cu foil before UV/ozone treatment and analyzed for the full width half maximum (FWHM) and I2D/IG intensity ratio. The observed FWHM of 2D band ranges from 34.4 to 61.5 cm1, the intensity ratio of I2D/IG ranges from 0.15 to 2.8, as shown in Figs. S1(a) and (b). As reported for Raman spectral analysis of bilayer graphene, the FWHM of 2D band and I2D/IG ratio of all spectra measured in this study correspond to either AB-stacked bilayer, disoriented-stacked or twisted bilayer graphene [19,26e28]. The sheet resistance of bilayer graphene transferred to a PET film was measured by noncontact measurements using eddy current method for focusing on the effects of the UV/ozone treatment [22]. The average sheet resistance of bilayer graphene before the UV/ozone treatment was 328 U, and that after 20 min of UV/ozone treatment was 364 U. The electrical conductively of the UV/ozonetreated bilayer graphene does not deteriorate markedly, and compared with GO and amorphous carbon membranes, it demonstrates a sufficient ability to suppress the charging during lowtemperature TEM observations. Fig. 2(a) shows a STEM image of bilayer graphene without the UV/ozone treatment. The 13
stacking misalignment was measured from the inset fast Fourier transform (FFT) pattern in Fig. 2(a).
pattern was clearly observed, Because of this misalignment, a Moire however, defects due to lack of carbon atoms were not confirmed. The effect of stacking misalignment on defect formation is not clear, however, in the case of bilayer graphene with 13
misalignment, defects lacking approximately 200 carbon atoms were formed by the UV/ozone treatment as shown in Fig. 2(b). Interestingly, the formation of defects was limited to the upper layer of bilayer graphene; no defects were observed in the underlying Monolayer region (dark region in the image) exposed by defect formation. To
pattern and defect area, atomic models of bilayer clarify the Moire graphene with 13
stacking misalignment without or with UV/ ozone treatment are shown in Fig. 2(c). The carbon sp2 bond cleaved by UV irradiation becomes an active dangling bond, and active oxygen radicals and hydrophilic groups are adsorbed there [23,24,29]. The defect area is considered to expand because of carbon desorption as carbon monoxide (CO) or carbon dioxide (CO2), which is molecularized by the oxidation reaction by oxygen functional groups [29]. However, in the lower layer, the carbon sp2 binding energy is much larger than the reaction-barrier energy of dangling-bond at the vacancy site [30], thus, UV/ozone treatment for 20 min produced no defects. As shown in Fig. S2, the effect of

UV/ozone treatment in this work is uniform across a hole of grid. And defects were observed only in the upper layer as well at any stacking angle in this study. That is, it is suggested that the uniformity of the effect of UV/ozone treatment is not impaired even with polycrystalline bilayer graphene. Using a constant of volume of the dropping solution, the thickness and surface area of the formed solution layer on the substrate depends greatly on the water contact angle according to
theory [31,32]. In other words, if the blotting proYoung-Dupre cesses are under the same conditions of the sample preparation, then it is suggested that the contact angle is greatly dependent on the control of the ice thickness. Fig. 3 shows the relationship between the UV/ozone treatment time and the water contact angle of bilayer graphene. As shown in Fig. 3, as UV/ozone treatment time increase, the water contact angle decreases After the UV irradiation time increase to 10 min, the angle reach to approximately 30
and saturates. The characteristics of the hydrophilization process by UV/ ozone treatment differ from those of the plasma treatment. One difference is the small number of introduced defects, as evident from the Raman measurements. A low defect density is beneficial for maintaining the high conductivity of graphene. The other difference is the wide range of hydrophilization degrees. Because the image of a biological sample by cryo-TEM is greatly affected by the thickness of the vitreous ice layer, the ice layer should be as thin as possible to attain a high-resolution image with a high signal-tonoise ratio. However, if the ice is too thin, the three-dimensional structure of the biological sample may be distorted by surface tension, resulting in the acquisition of inaccurate structural information. Therefore, the ice thickness should be optimized according to the target biological sample and to the acceleration voltage of the TEM. To confirm the applicability of UV/ozone-treated bilayer graphene grid to low-temperature TEM, we attempted to observe tobacco mosaic virus (TMV) in a buffer layer formed on the grid [33]. Imaged by a Cs-corrected, monochromator-equipped TEM at an acceleration voltage of 60 kV, several TMVs imaged at amazing high contrast in the very thin vitreous ice layer on the bilayer graphene (Fig. 4(a)). The periodic helical structure composed of subunits of capsomer proteins were clear, this is known to surround ribosomal nucleic acids. The thickness of the vitreous ice layer in which TMVs were embedded was measured by STEM-EELS. Assuming that the inelastic scattering probability increases exponentially with increasing the sample thickness [34], the following equation is given,

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Fig. 2. (a), (b) STEM image of bilayer graphene without and with UV/ozone treatment, the inset shows FFT patterns corresponding to each STEM images. (c) Atomic models based on the STEM images and FFT patterns in (a) and (b). (A colour version of this figure can be viewed online.)

,
2 1 þ E0 1022 1 þ E0 511 =

=


F¼

Em ¼ 7:6Zeff 0:36

(3)

(4)

where, b is the collection semi-angle of the microscope and Zeff is the effective atomic number of the sample. The effective atomic number of the heterogeneous sample is calculated by the following equation using the total number of electrons (f) and the atomic number (Z) of each element [36].

Zeff ¼

Fig. 3. Relationship between water contact angle on bilayer graphene surface and UV/ ozone treatment time, and examples of optical microscope images used to measure the angles. (A colour version of this figure can be viewed online.)


 t It ¼ ln L I0

(1)

where t is the sample thickness, L is the inelastic mean free path, and I0 and It represent the intensity of the zero-loss peak in EELS and that of the entire spectrum including the zero-loss peak, respectively. Furthermore, L, which is dependent on the acceleration voltage (E0) and the effective atomic mass of the sample (Em), is calculated by the following equations [35]:



ln 2bE0=E L ¼ 106F E0=E m m

(2)

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi f 1  ðZ1 Þ þ f 2  ðZ2 Þ þ f 3  ðZ3 Þ þ /

2:92

(5)

In this study, the acceleration voltage was 60 kV; thus, the mean free path L of inelastic scattering in the ice layer was calculated to be 52.9 nm. Fig. 4(c) is a low magnification STEM annular dark field (STEMADF) image of the ice formed on a Quantifoil hole covered with the UV/ozone-treated bilayer graphene. A typical spectrum of low-loss region taken from the hole of Fig. 4(c) is shown in Fig. 4(b), and its inset shows a fine spectrum of the 5e25 eV energy region. The peaks at 8.8, 10.6, and 14.5 eV are attributed to the transition from the valence band to the conduction band of ice [37,38]. Peaks at 17 and 21 eV are attributed to the surface and volume plasmon, respectively [39,40]. Some broad peaks in the fine spectrum suggest that an amorphous ice layer formed. Fig. 4(d) shows the ice layer thickness profile calculated on the basis of the spectral intensity ratio obtained with the white dashed line in Fig. 4(c). The average thickness of the ice layer is approximately 25 nm, which is thicker than the 18 nm diameter of TMV [33]. When the thickness of vitreous ice layers was measured using amorphous carbon such as a Quantifoil, lacey, or C-flat thick carbon grid, a thinner ice layer is known to be obtained by using a thin carbon film [41]. The thickness of the vitreous ice has also been reported to exhibit a smooth gradient from the center to the thicker end of the hole [41]. This smooth gradient is attributable to the

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Fig. 4. (a) TEM image of ice embedded TMVs on the UV/ozone treated-bilayer graphene. (b) Typical low-loss region spectrum of an ice layer, the inset shows the corresponding fine spectrum. (c) Low magnification STEM-ADF image of a Quantifoil hole with vitreous ice on UV/ozone treated-bilayer graphene. (d) Profile of vitreous ice thickness of the white dashed line in (c). (A colour version of this figure can be viewed online.)

difference in surface free energy between the carbon and vacuum parts, which hinders efficient data acquisition. Therefore, when UV/ ozone-treated bilayer graphene covers the entire surface of Quantifoil, the thinnest ice layer is formed on the free-standing bilayer graphene in the vacuum region, as shown in Fig. 4(d). This result is in good agreement with the tendency for thinner ice layers to form on thinner carbon layers [41]. Furthermore, because the surface free energy becomes more uniform the Quantifoil holes covered with bilayer graphene, a more homogeneous thin vitreous ice layer is formed as shown in Fig. 4(d). It also facilitates data acquisition and the following data processing. These results show that a more homogeneous thin vitreous ice layer can be formed by hydrophilizing the upper layer of bilayer graphene. The ultimate thinness and high electrical conductivity, known as the inherent characteristics of graphene, are effective in suppressing background noise and charge-up in TEM. By combining this feature with the UV/ozonehydrophilization technique of bilayer graphene, recent highperformance low-acceleration voltage TEM are expected to image smaller polymers, gels and soft-materials in frozen aqueous layer at a high signal-to-noise ratio. 4. Summary A new grid for cryo-TEM was developed using plasma-enhanced CVD grown bilayer graphene. High hydrophilicity was imparted by introducing defects only in the upper layer of the bilayer graphene via UV/ozone treatment. In this process, the crystallinity of the lower layer is maintained. Because this method results in a grid with a wide range of hydrophilicity, it can be applied to cryo-TEM of proteins of various sizes. A high signal-to-noise ratio cryo-TEM observation at low acceleration voltage of 60 kV was demonstrated using this grid, and the periodic helical structure of TMV

embedded in a very thin and homogeneous vitreous ice layer stably formed on this grid was demonstrated. These results indicate that the bilayer graphene TEM grid imparted with hydrophilicity by a UV/ozone treatment is very useful for cryo-TEM method for protein as well as other molecules including smaller polymers and soft materials. Author’s contributions R. K. and M. H. designed the experiments and prepared the base material. Y. H., N. K. and C. S. prepared the biological sample. R. K., Y. H., and N. K. performed cryo-TEM and EELS. R. K. performed TEM and STEM. R. K. and K. S. analyzed the data. R. K., C. S., and M. H. cowrote the paper. All of authors commented on the manuscript. Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbon.2020.01.010. References [1] H. Sjors, W. Scheres, A bayesian view on cryo-EM structure determination, J. Mol. Biol. 415 (2012) 406e418. [2] J. Frank, B. Shimkin, H. Dowse, Spider-A Modular software system for electron image processing, Ultramicroscopy 6 (1981) 343e358. [3] J. Dubochet, J.J. Chang, R. Freeman, J. Lepault, A.W. McDowall, Frozen aqueous suspensions, Ultramicroscopy 10 (1982) 55e61.

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