Microscopy, 2014, Vol. 63, No. S1
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Acknowledgment The author would like to thank Dr. Ichirou Karahara (Univ. Toyama), Dr. L. Andrew Staehelin (Univ. Colorado), Ms. Naoko Kajimura, Dr. Akio Takaoka (Osaka Univ.), Dr. Kazuyo Misaki, Dr. Shigenobu Yonemura (RIKEN CDB), Dr. Kazuyoshi Murata (NIP), Dr. Kentaro Uesugi, Dr. Akihisa Takeuchi, Dr. Yoshio Suzuki (JASRI), Dr. Miyuki Takeuchi, Dr. Daisuke Tamaoki, Dr. Daisuke Yamauchi, and Ms. Aki Fukuda (Univ. Hyogo) for their collaborations in the work presented here. References 1. Murata, et al. (2002) J. Electron Microsc. 51, 133–136; 2. Karahara, et al. (2009) Plant J. 57, 819–831; 3. Karahara, et al. (2012) In “Molecular regulation of endocytosis” Ceresa B. ed., InTech, pp. 41–60; 4. Yamauchi, et al. (2012) AIP Conf. Proc. 1466, 237–242; 5. Yamauchi, et al. (2013) Microscopy 62, 353–36 doi: 10.1093/jmicro/dfu036 3D structure determination of protein using TEM single particle analysis Chikara Sato, Kazuhiro Mio, Masaaki Kawata, and Toshihiko Ogura National Institute of Advanced Industrial Science and Technology (AIST) Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone
secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1–5] and applied them to different proteins [6–8]. Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease-type. Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. The structure of human SPP was determined by SPA at a resolution of 22 Å [8]. SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 x 85 x 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. For the tetrameric assembly, the N-terminal region of SPP was found to be sufficient. Moreover, when N-terminal region was overexpressed, the formation of the endogenous SPP tetramer was inhibited, which suppressed the proteolytic activity within cells. From these data, the N-terminal region is considered to work as the structural scaffold. Transmembrane (TM) translocation of newly synthesized secretion proteins and membrane proteins are carried out by a Sec translocon protein complex. The polypeptide-conducting pore is formed by the SecYEG-SecA complex in bacteria, and the membrane protein SecDF is necessary for the efficient transport of proteins. However the molecular mechanism how SecDF realized efficient transport is not clear. A previous X-ray structural study of the whole protein and subdomain suggest that SecDF has at least two conformational variants, which could reflect molecular dynamics of this protein. To confirm this hypothesis, we analyzed the 3D structure of SecDF using dark field STEM electron tomography and single particle reconstruction. We determined two different whole SecDF protein structures which well explains the X-ray data. From these data, we would like to propose the possible molecular mechanism of SecDF during polypeptide translocation.
References 1. Ogura T, Sato C (2001) An automatic particle pickup method using a neural network applicable to low-contrast electron micrographs. J. Struct. Biol. 136: 227–238. 2. Ogura T, Iwasaki T, Sato C (2003) Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking. J. Struct. Biol. 143: 185–200. 3. Ogura T, Sato C (2006) A fully automatic 3D reconstruction method using simulated annealing enables accurate posterioric angular assignment of protein projections. J. Struct. Biol. 156: 371–386.
Downloaded from http://jmicro.oxfordjournals.org/ at Memorial University of Newfoundland on March 5, 2015
established during cell division is an essential question in plant morphogenesis. Herein we demonstrate how computer tomography techniques can aid in understanding nano-machines involved in determination of the division site and in analysing air space development after cytokinesis. The preprophase band (PPB) is a cytokinetic nano-machine used to determine the plant division site. The PPB appears as a broad microtubule (MT) band in the G2 phase and the MT band narrows during the prophase to establish the specialized belt zone in the cell cortex (cortical division zone, [CDZ]). The MT band disappears at prometaphase, but some memories remain in the CDZ throughout the process of cell division, and this is the site of attachment of the newly formed cell plate. We have examined PPB development of high-pressure frozen onion cotyledon epidermis using dual-axis electron tomography. MTs as well as actin microfilaments (MFs) and membrane systems can be preserved well by high-pressure freezing [1]. Since detection of ∼100 vesicles and ∼40 MT ends was possible in a tomogram of the PPB surface (0.25 mm × 0.25 mm) obtained from 250-nm-thick tangential sections of epidermal cells, we were able to quantitatively analyze the frequencies of various types of vesicles and MT ends in the PPB [2]. The results clearly showed that endocytosis is active [2,3] and MTs are very dynamic in the late PPB. Light microscopic studies with fluorescent probes have demonstrated that actins are among the main components of PPB. Electron tomography analysis showed that one actin configuration in the PPB is a relatively short single MFs running parallel to the plasma membrane. The actin MFs connecting two adjacent MTs help to promote MT bundling. Cell plate attachment to the parental wall leads to the fusion of the newly formed middle lamellae in the cell plate to the middle lamella of parental cell wall, and a three-way junction is created. Air space develops from the three-way junction. To determine 3D arrangement of cells and air spaces, we used X-ray micro-CT at the SPring-8 synchrotron radiation facility. Using micro-CT available in BL20XU (8 keV, 0.2 µm/pixel), we were able to elucidate ∼90% of the cortical cell outlines in the hypocotyl-radicle axis of arabidopsis seeds [4] and to analyze cell geometrical properties. As the strength of the system X-ray is too strong for seed survival, we used another beam line BL20B2 (10-15 keV, 2.4-2.7 µm/pixel) to examine air space development during seed imbibition [4,5]. Using this system, we were able to detect air space development at the early imbibition stages of seeds without causing damage during seed germination.
Microscopy, 2014, Vol. 63, No. S1
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doi: 10.1093/jmicro/dfu074
Near-infrared nano-imaging spectroscopy using a phase change mask method Yu Sato, Shohei Kanazawa, and Toshiharu Saiki Department of Electronics and Electrical Engineering, Keio University 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan We propose a technique that employs an optical mask layer of a phase-change material, e.g. GeSbTe, which is widely used for rewritable optical recording media, for realizing highly sensitive near-field imaging spectroscopy of single semiconductor quantum constituents at optical telecommunication wavelengths. Semiconductor quantum dots (QDs) have shown great promise as efficient single photon emitters and entangled photon sources, making them attractive for quantum communication and quantum information processing applications. Self-assembled InAs QDs on InP substrate are promising as near-infrared (NIR) single photon and entangled photon emitters. In order to clarify and control the optical properties of QDs for telecommunication devices, photoluminescence (PL) spectroscopy studies of single QDs with high spatial resolution at NIR wavelength is necessary. The most useful technique to attain this is by using near-field scanning optical miscroscopy (NSOM). However, NSOM has a lower PL collection efficiency at NIR wavelength than at visible wavelength [1]. This problem inhibits NIR-PL spectroscopy based on NSOM to be practically realized. Therefore, we deveopled a method to overcome the low NIR-PL spectroscopy by using a nanoaperture on an optical mask layer of phase-change material (PCM) [2]. Due to the large optical contrast between the crystalline and amorphous phases of the phase-change material at visible wavelengths and its high transparency at NIR wavelengths, an amorphous nanoaperture can be used to realize imaging spectroscopy with a high spatial resolution and a high collection efficiency (Fig. 1). We demonstrate the effectiveness of the proposed method by performing numerical simulations and PL measurements of InAs/InP QDs.
Fig. 1. Schematic illustration of phase change mask method
PCM mask effect has also the potential to be applied in emission energy control of QDs. One of the main problems for realization of quantum communication applications is precise control of energy in QDs. We proposed a new approach to control the emission energy of QDs by applying a local strain using volume expansion of phasechange material [3–5]. We calculated the stress and energy shift distribution induced by volume expansion using finite element method. Simulation result reveals that redshift is obtained beneath the flat part of amorphous mark, while blueshift is obtained beneath the edge region of amorphous mark. Simulation result is accompanied by two experimental studies; two-dimensional PL intensity mapping of InAs/ InP QD sample deposited by a layer of PCM, and an analysis on the relationship between PL intensity ratio and energy shift were performed.
References 1. Tsumori N., Takahashi M., Saiki T., Sakuma Y., Appl. Opt. 50, 5710 (2011). 2. Tsumori N., Takahashi M., Kubota R., Saiki T., Regreny P., Gendry M., Appl. Phys. Lett. 100, 063111 (2012). 3. Takahashi M., Syafawati Humam Nurrul, Tsumori N., Saiki T., Regreny P., Gendry M., Appl. Phys. Lett. 102, 093120 (2013). 4. Tsumori N., Takahashi M., Syafawati Humam Nurrul, Regreny P., Gendry M., Saiki T., Journal of Physics: Conference Series 471, 012007 (2013). 5. Nurrul Syafawati Binti Humam, Sato Y., Takahashi M., Kanazawa S., Tsumori N., Regreny P., Gendry M., Saiki T., Opt. Expr. 22, 14830 (2014). doi: 10.1093/jmicro/dfu089
High-resolution Structural Analysis on Ionic-Liquid/Solid Interfaces by Frequency Modulation Atomic Force Microscopy Takashi Ichii and Hiroyuki Sugimura Department of Materials Science and Engineering, Kyoto University,
E-mail: [email protected] Ionic liquids (ILs) are salts usually consisting of sterically-hindered organic/inorganic ions with melting points below 100 ○C or room temperature. Since they have a number of distinct physical and chemical properties, such as high ionic conductivity, high electrochemical stability, non-volatility, and non-combustibility, they have attracted considerable attention as a replacement for water and organic solvent. The interfaces between ILs and solid substrates play an important role in various applications including electrochemistry, heterogeneous catalysis, and lubrication, and thus, structural analysis on IL/solid interfaces would be both academically interesting and practically important. Frequency Modulation Atomic Force Microscopy (AFM) is known to be capable of atomic-scale imaging on solid substrates in various environments such as vacuum, atmosphere, and liquid. FM-AFM can be also applicable for investigating the density distribution of liquid molecules on liquid/solid interfaces and hence, it is also expected to provide helpful information for improving the IL-based applications mentioned above. Here, we report FM-AFM studies on IL/solid interfaces. A quartz tuning fork (QTF) sensor was used as a force sensor instead of a Si cantilever because the Q factor of a Si cantilever would be heavily suppressed in ILs. The Q factor of the QTF sensor was typically 100 in ILs, which was much higher than the reported Q factor of a Si cantilever in liquid environment. Figure 1(a) shows a topographic image of a KCl(100) surface obtained in 1-butyl-1-methylpyrrolidinium tris ( pentafluoroethyl)trifluorophosphate ([Py1,4]FAP). While the viscosity of the IL was as approximately 400 times high as that of water (346 cP), the square-lattice structure with a period of ∼0.4 nm was clearly imaged. That is, atomic-resolution imaging was successfully
Downloaded from http://jmicro.oxfordjournals.org/ at Memorial University of Newfoundland on March 5, 2015
4. Kawata M, Sato C (2007) A statistically harmonized alignmentclassification in image space enables accurate and robust alignment of noisy images in single particle analysis. J. Electron Microsc. 56: 83–92. 5. Kawata M, Sato C (2013) Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images. Microscopy 62: 303–315. 6. Ogura T, Tong KI, Mio K, Maruyama Y, Kurokawa H, Sato C, Yamamoto M (2010) Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains. Proc Natl Acad Sci U S A. 107:2842–2847. 7. Yajima H, Ogura T, Nitta R, Okada Y, Sato C, Hirokawa N (2012) Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy. J Cell Biol. 198: 315–322. 8. Miyashita H, Maruyama Y, Isshiki H, Osawa S, Ogura T, Mio K, Sato C, Tomita T, Iwatsubo T (2011) Three-dimensional structure of the signal peptide peptidase. J Biol Chem. 286: 26188–26197.
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Acknowledgment The author would like to thank Dr. Ichirou Karahara (Univ. Toyama), Dr. L. Andrew Staehelin (Univ. Colorado), Ms. Naoko Kajimura, Dr. Akio Takaoka (Osaka Univ.), Dr. Kazuyo Misaki, Dr. Shigenobu Yonemura (RIKEN CDB), Dr. Kazuyoshi Murata (NIP), Dr. Kentaro Uesugi, Dr. Akihisa Takeuchi, Dr. Yoshio Suzuki (JASRI), Dr. Miyuki Takeuchi, Dr. Daisuke Tamaoki, Dr. Daisuke Yamauchi, and Ms. Aki Fukuda (Univ. Hyogo) for their collaborations in the work presented here. References 1. Murata, et al. (2002) J. Electron Microsc. 51, 133–136; 2. Karahara, et al. (2009) Plant J. 57, 819–831; 3. Karahara, et al. (2012) In “Molecular regulation of endocytosis” Ceresa B. ed., InTech, pp. 41–60; 4. Yamauchi, et al. (2012) AIP Conf. Proc. 1466, 237–242; 5. Yamauchi, et al. (2013) Microscopy 62, 353–36 doi: 10.1093/jmicro/dfu036 3D structure determination of protein using TEM single particle analysis Chikara Sato, Kazuhiro Mio, Masaaki Kawata, and Toshihiko Ogura National Institute of Advanced Industrial Science and Technology (AIST) Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone
secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1–5] and applied them to different proteins [6–8]. Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease-type. Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. The structure of human SPP was determined by SPA at a resolution of 22 Å [8]. SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 x 85 x 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. For the tetrameric assembly, the N-terminal region of SPP was found to be sufficient. Moreover, when N-terminal region was overexpressed, the formation of the endogenous SPP tetramer was inhibited, which suppressed the proteolytic activity within cells. From these data, the N-terminal region is considered to work as the structural scaffold. Transmembrane (TM) translocation of newly synthesized secretion proteins and membrane proteins are carried out by a Sec translocon protein complex. The polypeptide-conducting pore is formed by the SecYEG-SecA complex in bacteria, and the membrane protein SecDF is necessary for the efficient transport of proteins. However the molecular mechanism how SecDF realized efficient transport is not clear. A previous X-ray structural study of the whole protein and subdomain suggest that SecDF has at least two conformational variants, which could reflect molecular dynamics of this protein. To confirm this hypothesis, we analyzed the 3D structure of SecDF using dark field STEM electron tomography and single particle reconstruction. We determined two different whole SecDF protein structures which well explains the X-ray data. From these data, we would like to propose the possible molecular mechanism of SecDF during polypeptide translocation.
References 1. Ogura T, Sato C (2001) An automatic particle pickup method using a neural network applicable to low-contrast electron micrographs. J. Struct. Biol. 136: 227–238. 2. Ogura T, Iwasaki T, Sato C (2003) Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking. J. Struct. Biol. 143: 185–200. 3. Ogura T, Sato C (2006) A fully automatic 3D reconstruction method using simulated annealing enables accurate posterioric angular assignment of protein projections. J. Struct. Biol. 156: 371–386.
Downloaded from http://jmicro.oxfordjournals.org/ at Memorial University of Newfoundland on March 5, 2015
established during cell division is an essential question in plant morphogenesis. Herein we demonstrate how computer tomography techniques can aid in understanding nano-machines involved in determination of the division site and in analysing air space development after cytokinesis. The preprophase band (PPB) is a cytokinetic nano-machine used to determine the plant division site. The PPB appears as a broad microtubule (MT) band in the G2 phase and the MT band narrows during the prophase to establish the specialized belt zone in the cell cortex (cortical division zone, [CDZ]). The MT band disappears at prometaphase, but some memories remain in the CDZ throughout the process of cell division, and this is the site of attachment of the newly formed cell plate. We have examined PPB development of high-pressure frozen onion cotyledon epidermis using dual-axis electron tomography. MTs as well as actin microfilaments (MFs) and membrane systems can be preserved well by high-pressure freezing [1]. Since detection of ∼100 vesicles and ∼40 MT ends was possible in a tomogram of the PPB surface (0.25 mm × 0.25 mm) obtained from 250-nm-thick tangential sections of epidermal cells, we were able to quantitatively analyze the frequencies of various types of vesicles and MT ends in the PPB [2]. The results clearly showed that endocytosis is active [2,3] and MTs are very dynamic in the late PPB. Light microscopic studies with fluorescent probes have demonstrated that actins are among the main components of PPB. Electron tomography analysis showed that one actin configuration in the PPB is a relatively short single MFs running parallel to the plasma membrane. The actin MFs connecting two adjacent MTs help to promote MT bundling. Cell plate attachment to the parental wall leads to the fusion of the newly formed middle lamellae in the cell plate to the middle lamella of parental cell wall, and a three-way junction is created. Air space develops from the three-way junction. To determine 3D arrangement of cells and air spaces, we used X-ray micro-CT at the SPring-8 synchrotron radiation facility. Using micro-CT available in BL20XU (8 keV, 0.2 µm/pixel), we were able to elucidate ∼90% of the cortical cell outlines in the hypocotyl-radicle axis of arabidopsis seeds [4] and to analyze cell geometrical properties. As the strength of the system X-ray is too strong for seed survival, we used another beam line BL20B2 (10-15 keV, 2.4-2.7 µm/pixel) to examine air space development during seed imbibition [4,5]. Using this system, we were able to detect air space development at the early imbibition stages of seeds without causing damage during seed germination.
Microscopy, 2014, Vol. 63, No. S1
i10
doi: 10.1093/jmicro/dfu074
Near-infrared nano-imaging spectroscopy using a phase change mask method Yu Sato, Shohei Kanazawa, and Toshiharu Saiki Department of Electronics and Electrical Engineering, Keio University 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan We propose a technique that employs an optical mask layer of a phase-change material, e.g. GeSbTe, which is widely used for rewritable optical recording media, for realizing highly sensitive near-field imaging spectroscopy of single semiconductor quantum constituents at optical telecommunication wavelengths. Semiconductor quantum dots (QDs) have shown great promise as efficient single photon emitters and entangled photon sources, making them attractive for quantum communication and quantum information processing applications. Self-assembled InAs QDs on InP substrate are promising as near-infrared (NIR) single photon and entangled photon emitters. In order to clarify and control the optical properties of QDs for telecommunication devices, photoluminescence (PL) spectroscopy studies of single QDs with high spatial resolution at NIR wavelength is necessary. The most useful technique to attain this is by using near-field scanning optical miscroscopy (NSOM). However, NSOM has a lower PL collection efficiency at NIR wavelength than at visible wavelength [1]. This problem inhibits NIR-PL spectroscopy based on NSOM to be practically realized. Therefore, we deveopled a method to overcome the low NIR-PL spectroscopy by using a nanoaperture on an optical mask layer of phase-change material (PCM) [2]. Due to the large optical contrast between the crystalline and amorphous phases of the phase-change material at visible wavelengths and its high transparency at NIR wavelengths, an amorphous nanoaperture can be used to realize imaging spectroscopy with a high spatial resolution and a high collection efficiency (Fig. 1). We demonstrate the effectiveness of the proposed method by performing numerical simulations and PL measurements of InAs/InP QDs.
Fig. 1. Schematic illustration of phase change mask method
PCM mask effect has also the potential to be applied in emission energy control of QDs. One of the main problems for realization of quantum communication applications is precise control of energy in QDs. We proposed a new approach to control the emission energy of QDs by applying a local strain using volume expansion of phasechange material [3–5]. We calculated the stress and energy shift distribution induced by volume expansion using finite element method. Simulation result reveals that redshift is obtained beneath the flat part of amorphous mark, while blueshift is obtained beneath the edge region of amorphous mark. Simulation result is accompanied by two experimental studies; two-dimensional PL intensity mapping of InAs/ InP QD sample deposited by a layer of PCM, and an analysis on the relationship between PL intensity ratio and energy shift were performed.
References 1. Tsumori N., Takahashi M., Saiki T., Sakuma Y., Appl. Opt. 50, 5710 (2011). 2. Tsumori N., Takahashi M., Kubota R., Saiki T., Regreny P., Gendry M., Appl. Phys. Lett. 100, 063111 (2012). 3. Takahashi M., Syafawati Humam Nurrul, Tsumori N., Saiki T., Regreny P., Gendry M., Appl. Phys. Lett. 102, 093120 (2013). 4. Tsumori N., Takahashi M., Syafawati Humam Nurrul, Regreny P., Gendry M., Saiki T., Journal of Physics: Conference Series 471, 012007 (2013). 5. Nurrul Syafawati Binti Humam, Sato Y., Takahashi M., Kanazawa S., Tsumori N., Regreny P., Gendry M., Saiki T., Opt. Expr. 22, 14830 (2014). doi: 10.1093/jmicro/dfu089
High-resolution Structural Analysis on Ionic-Liquid/Solid Interfaces by Frequency Modulation Atomic Force Microscopy Takashi Ichii and Hiroyuki Sugimura Department of Materials Science and Engineering, Kyoto University,
E-mail: [email protected] Ionic liquids (ILs) are salts usually consisting of sterically-hindered organic/inorganic ions with melting points below 100 ○C or room temperature. Since they have a number of distinct physical and chemical properties, such as high ionic conductivity, high electrochemical stability, non-volatility, and non-combustibility, they have attracted considerable attention as a replacement for water and organic solvent. The interfaces between ILs and solid substrates play an important role in various applications including electrochemistry, heterogeneous catalysis, and lubrication, and thus, structural analysis on IL/solid interfaces would be both academically interesting and practically important. Frequency Modulation Atomic Force Microscopy (AFM) is known to be capable of atomic-scale imaging on solid substrates in various environments such as vacuum, atmosphere, and liquid. FM-AFM can be also applicable for investigating the density distribution of liquid molecules on liquid/solid interfaces and hence, it is also expected to provide helpful information for improving the IL-based applications mentioned above. Here, we report FM-AFM studies on IL/solid interfaces. A quartz tuning fork (QTF) sensor was used as a force sensor instead of a Si cantilever because the Q factor of a Si cantilever would be heavily suppressed in ILs. The Q factor of the QTF sensor was typically 100 in ILs, which was much higher than the reported Q factor of a Si cantilever in liquid environment. Figure 1(a) shows a topographic image of a KCl(100) surface obtained in 1-butyl-1-methylpyrrolidinium tris ( pentafluoroethyl)trifluorophosphate ([Py1,4]FAP). While the viscosity of the IL was as approximately 400 times high as that of water (346 cP), the square-lattice structure with a period of ∼0.4 nm was clearly imaged. That is, atomic-resolution imaging was successfully
Downloaded from http://jmicro.oxfordjournals.org/ at Memorial University of Newfoundland on March 5, 2015
4. Kawata M, Sato C (2007) A statistically harmonized alignmentclassification in image space enables accurate and robust alignment of noisy images in single particle analysis. J. Electron Microsc. 56: 83–92. 5. Kawata M, Sato C (2013) Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images. Microscopy 62: 303–315. 6. Ogura T, Tong KI, Mio K, Maruyama Y, Kurokawa H, Sato C, Yamamoto M (2010) Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains. Proc Natl Acad Sci U S A. 107:2842–2847. 7. Yajima H, Ogura T, Nitta R, Okada Y, Sato C, Hirokawa N (2012) Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy. J Cell Biol. 198: 315–322. 8. Miyashita H, Maruyama Y, Isshiki H, Osawa S, Ogura T, Mio K, Sato C, Tomita T, Iwatsubo T (2011) Three-dimensional structure of the signal peptide peptidase. J Biol Chem. 286: 26188–26197.
