Controlled Synthesis of Gold Nanoparticles on Fluorescent

National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. ACS Appl. Nano Mater. , Articl...
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Controlled Synthesis of Gold Nanoparticles on Fluorescent Nanodiamond via Electron Beam-Induced Reduction Method for Dualmodal Optical and Electron Bioimaging Masahito Morita, Takashi Tachikawa, Satoshi Seino, Koji Tanaka, and Tetsuro Majima ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00213 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Controlled Synthesis of Gold Nanoparticles on Fluorescent Nanodiamond via Electron BeamInduced Reduction Method for Dualmodal Optical and Electron Bioimaging Masahito Morita,*,† Takashi Tachikawa,‡ Satoshi Seino,§ Koji Tanaka,⊥ and Tetsuro Majima,*,‡ †

WPI Immunology Frontier Research Center, (iFReC), Osaka University, 3-1 Yamadaoka, Suita,

Osaka 567-0871, Japan ‡

The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki,

Osaka 567-0047, Japan §



Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan Research Institute of Electrochemical Energy, National Institute of Advanced Industrial

Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

Keywords: multimodal imaging, fluorescent nanodiamond, gold nanoparticles, hybrid nanoparticles, plasmonic modulation, fluorescence lifetime imaging, element-selective imaging, quantum beam

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ABSTRACT Hybrid nanoparticles are emergent nanomaterials that combine particles with different characteristic properties to enhance their original functions or modulate their original physical or chemical properties for application in catalysis, sensing, and imaging. Fluorescent nanodiamonds (fNDs) have recently become more attractive for bioimaging due to their characteristic physicochemical properties and biocompatibility. Their wide applicability in bioimaging has been utilized in the single-particle tracking of biomolecules, local environmental sensors in cells, and stem cell tracking in tissues. However, the use of fNDs as multiscale spatial mapping probes for multiple biomolecules and cells in optical and electron microscopy techniques has been limited because of their broad fluorescence spectrum and composition of mainly light elements (C, O, H, N, etc.), respectively. On the other hand, metal nanoparticles (metal NPs) with unique photonic properties have been employed as functional labelling probes in bioimaging. Therefore, an efficient synthesis strategy to produce fND/metal NP nanocomposites with regulated shapes is required to develop molecular and cellular bioimaging probes with simultaneous use in multiple imaging techniques. Here, we report the synthesis of dualmodal hybrid gold NP-fND (Au-NDs) nanoparticles with a mean diameter of less than 20 nm using an electron beam-induced reduction method. The resultant Au-NDs exhibited stable Au NP-induced plasmonic modulation of fluorescence lifetimes in cellular environments, which is useful for fluorescence lifetime imaging microscopy to detect multiple molecules or cells. Furthermore, Au NPs modified on fNDs function as surrogate markers with sub-10 nm spatial resolution for electron microscopy in mammalian cells. Our findings indicate that the electron-beam reduction method will enable us to make simplified formations of metal NPs with characteristic plasmonic structures on fNDs for multimodal bioimaging probes.

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INTRODUCTION In the life sciences, including in the biomedical and cell biological fields, bioimaging with multiple modalities (light, electron, magnetic field, radiation, acoustic wave, etc.), which has been recently termed multimodal imaging, has been used to evaluate the hierarchical distribution and physiological function of multiple labeled biosubstrates within cells or administrated cells in tissues.1-4 For the region between the nm and µm scales, biological transmission electron microscopy (TEM) and fluorescence microscopy are suitable imaging tools and have been frequently used as dualmodal imaging tools to simultaneously analyze the distribution and dynamic behaviors of several kinds of biomolecules and cells.5,6 For this technique, dualmodal imaging probes have been developed.7-9 In principle, dualmodal imaging probes should have low toxicity and high stability for a long term to function as molecular cellular labelling probes. Nanodiamonds (NDs) have received considerable attention in the fields of bioimaging, magnetic sensing, and temperature monitoring and in drug delivery systems because of their extraordinary physical and chemical properties, biocompatibility, and a multitude of potential applications.10-20 In particular, the nitrogen-vacancy (NV) defect centers in NDs (200 nm), and a long fluorescence lifetime (>10 ns).21-23 Therefore, as long-term stable markers in the field of regenerative medicine and immunotherapy, NDs with these characteristic properties are suitable for monitoring the distribution and movement of biomolecules or drugs and local environmental factors such as temperature and electrical potential in cells or transplanted cells in tissues.24-29 In this sense, NDs are alternative candidates for dualmodal imaging probes.30 However, they have

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several limitations. The wide fluorescence spectrum hampers the simultaneous multiple labeling of biomolecules or cells, unlike organic dyes and quantum dots, which produce sharp emission spectra and a variety of emission wavelengths. Furthermore, it is difficult to determine their precise locations within cells by biological TEM because of the weak contrast of light elements (C, O, H, N, etc.) in the NDs and their sizes are still larger than those of cellular proteins and assembled molecular machinery such as ribosome and membrane proteins. To overcome these limitations of NDs as dualmodal imaging probes, it is necessary to modulate their optical properties, reduce the mean diameter of NDs to less than several tens of nanometers, and attach high-contrast TEM probes with large element numbers onto the NDs. Recently, nanocomposites of NDs with metal nanoparticles (NPs), especially gold NPs (Au NPs) have emerged as attractive hybrid nanomaterials for biological and materials fields, including in bioimaging probes, catalysis, and electric devices.31-37 These metal NPs with characteristic plasmonic bands are highly biocompatible and have been employed to modulate the optical properties of adsorbed chromophores. Additionally, Au NPs have been used as immunogold labelling tools for the visualization of labeled biomolecules with nm-scale spatial resolution in biological TEM.38 An advantage of using such metal NPs is the plasmonic phenomenon to enhance the optical properties of NV centers, which significantly govern the fluorescence intensity and lifetime.39 For these nanocomposites, the plasmonic modulation of the fluorescence lifetime of NV centers through interaction with Au NPs enables us to monitor different biomolecules or cells during fluorescence lifetime imaging (FLIM), and the metals on Au-NDs act as probes to monitor the Au-NDs in biological TEM.40 The modulation of the fluorescence lifetime depends on the size of the metal NPs and the distance between the NV centers and the metal NPs. In addition, the structures of nanocomposites have been known to be

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critical for the efficiency of penetration into cells or labelling of biomolecules without affecting their physiological functions. Therefore, uniform and precise control of the overall dimension of the hybrid materials is required for dualmodal bioimaging. Hence, the hybrid NPs of fluorescent NDs (fNDs) and Au NPs with a mean diameter of less than 20 nm must be suitable for dualmodal imaging probes in dualmodal optical and electron bioimaging. Previous reports demonstrated several bottom-up synthetic techniques for fabricating nanocomposites including the Fenton reaction-based formation of NPs on NDs and the chemical reduction of metal ions adsorbed on polymers attached on surface-modified NDs.31,32 However, it is necessary to maintain the hybrid Au-NDs as dissociated particles to label the proteins and cells efficiently. Thus, a simple and precise synthetic route to metal NP-fND nanocomposites with a mean diameter of less than 20 nm is worth investigating to suppress the aggregation of nanocomposites by reducing the numbers of synthetic steps involved. The electron-beam reduction method has been known to synthesize highly dispersed metal NPs in liquid solutions simply by electronbeam radiation for several seconds.42 Herein we describe a synthetic method for preparing Au-NDs using this simple electronbeam reduction technique. We found that Au NPs deposited on the surface of NDs to have the potential to visualize the NV centers of ND in living cells using FLIM microscopy as multiple cell labelling probes and surrogate markers of NDs with Au-element mapping within cells in electron microscopy.

RESULTS AND DISCUSSION To generate fluorescent NDs, we implanted helium ions (He+) into ND particles (mean diameter: ~10 nm) on silicon wafers (Scheme 1 and Figure S1(a)). Then, the He+-implanted NDs

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(HeNDs) were annealed at 1073 K for 2 h to activate defect structures. To remove the surface graphitic components and modify the surface functional groups, we treated the irradiated ND samples to be oxidized in air at 773 K for 5 h, followed by treatment in mixed strong acidic solutions at 365 K for 4 h. To synthesize Au NPs on HeNDs, the dispersed HeND solutions containing polyvinyl alcohol (PVA) and gold ions were irradiated by electron beams. The electron beam irradiation, which lasted only a few seconds in the liquid phase, efficiently formed Au NPs on HeND surfaces owing to reduction of Au ions.

Scheme 1. Synthesis of Au NP-fND nanocomposites by ion implantation and electron-beam irradiation.

In the electron-beam reduction process, several radical species including H• radicals, hydrated electrons, e−, and carbon radicals are generated as strong reductants during radiolysis of the water and PVA mixture, causing the reduction of Au ions to form Au NPs.43 In this study, the electron beam reduction method is further applied to the synthesis of nanocomposites of Au NPs with HeNDs, implying that a similar reduction mechanism is involved to form Au NPs followed by the attachment of An NPs on the surface of HeNDs (Figure S1(b)).44 In this case, the dispersed PVA in HeND solutions can control the diameter of Au NPs, leading to the formation of Au-NDs with a mean diameter of less than 20 nm.

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TEM analysis showed that a few Au NPs (mean number: 3.1 ± 2.2 (n = 37)) with diameters of 1.5–5.0 nm (mean diameter: 3.1 ± 0.7 nm (n = 116)) were attached on HeNDs (Figure 1(a)) to form as Au-NDs (mean diameter: 16.3 ± 5.7 nm (n = 88)). Few HeNDs without Au NPs could also be found. High-resolution TEM analysis showed that both HeNDs and synthesized Au NPs had highly crystalline structures and Au NPs were deposited on the surface of HeNDs (Figure 1(b)). The absorption spectrum of Au-NDs showed a characteristic plasmon peak at around 510 nm, which also indicated the crystalline properties of Au NPs (Figure S2). Figure 1(c) shows that the high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and STEM-energy dispersive X-ray spectroscopy (STEM-EDX) images clearly visualize Au NPs as bright spots on dimmed regions, which are considered to be surrogate markers of NDs (Figures 1(c) and S3(a)). Therefore, electron microscopy imaging techniques can be used as background-free NDs tracking tools with sub-nm-level spatial resolution in cells.

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Figure 1. (a) TEM image of Au-NDs and the size distribution of Au-NDs; scale bar: 20 nm. (b) Atomic-resolution TEM image of a single Au-ND; scale bar: 2 nm. (c) HAADF-STEM image (1) and STEM-EDX spectrum image of each element (2: Au (red) + C (green), 3: Au, and 4: C map); scale bar: 10 nm.

In order to examine the fluorescence properties of HeNDs and Au-NDs under the microscope, we performed single-particle fluorescence analysis of the samples coated on cover glasses.45-47 Atomic force microscopy (AFM) data showed that the size distributions of the particles were similar to those obtained by TEM analysis (Figures S4(a), (b), (d), and (e)). To investigate the effects of Au NPs on the fluorescence properties of the NV center of HeNDs, we evaluated three optical characteristics: the fluorescence lifetime (τf), fluorescence intensity (If), and fluorescence spectrum. Figure 2(a) shows the typical fluorescence decay curves observed for

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single HeND and Au-ND particles. The decay profiles were well-fitted by three exponential functions. From the statistics of distributions of If against the weighted average τf for individual particles, the mean fluorescence lifetime () of HeNDs was determined to be 28.6 ± 12.9 ns (n: numbers of particles analyzed, n = 160), whereas = 10.3 ± 6.0 ns (n = 173) for Au-NDs. Since electron beam irradiation in HeND solutions did not change the value of (30.0 ± 15.5 ns (n = 148)) for HeNDs, the shortening of for Au-NDs is attributed to the surface modification of Au NPs (Figure 2(b)). Fluorescence decay analysis revealed that all three fitted parameters had shortened, implying that the plasmonic modulation of NV centers occurred because of Au NPs (Table 1). The If of each particle had slightly weakened. The If of Au-NDs was 1.80 ± 0.99 photons/ms (n = 173), whereas that of HeNDs was 3.36 ± 1.83 photons/ms (n = 148). We found no significant difference between the spectral shapes for two samples (Figures S4(c) and (f)). Each NP had a zero-phonon line at 575 nm, attributed to the NV0 state.48 Overall, the analyses indicate that Au-NDs have the potential to act as dualmodal imaging probes. Here, we discuss the mechanism of the optical modulation of the NV center by Au NPs on HeNDs. In general, two principle factors (radiative and non-radiative processes) affect the fluorescence properties (If and τf) of a chromophore (NV centers in this case) through metal–chromophore interactions, strongly depending on the distance and anisotropy between a chromophore and NPs and the diameter of Au NPs (Figure S5).49 When the distance between a chromophore and metal NPs is short, non-radiative processes via energy transfer from the excited states of the NV center to Au NPs are dominant, thus decreasing both the τf and If (i.e., fluorescence quantum yield) values. It was recently suggested that the lifetime of the quantum emitter is governed by the dynamics of the plasmons driven through dipole-dipole interactions.50 On the other hand, radiative processes can induce a fluorescence enhancement when the distance between a

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chromophore and Au NPs increases to cover the area of the NV centers. In this case, alignment of the emitting dipole of NV center with the propagation mode of the plasmon is also important. TEM analysis revealed that the localization of attachment of Au NPs on HeNDs was random on HeNDs and the mean diameter of Au NPs was smaller than 5 nm. Additionally, the localization of the NV centers depends on the size of HeNDs. Therefore, the distance between the NV centers and Au NPs ranged from the sub-nm scale to approximately 30 nm at most. The fluorescence of the proximal NV center near the interface between Au NPs and HeNDs might be quenched, whereas the enhancement in the radiative rate results in increase and decrease in the If and τf values, respectively. In this study, we showed that the attachment of Au NPs significantly shortens the τf of the NV center in Au-NDs and decreases the If, implying that the non-radiative process would be dominant. However, the quenching effect is not significant for simultaneous use of both particles (HeNDs and Au-NDs) to monitor multiple molecules or cells in the future. Although further studies are needed to understand the underlying mechanisms, Au NP-induced quenching of τf allows an application of Au-NDs as a biolabeling probe for the τf mapping, as demonstrated below.

Table 1. Average fluorescence lifetimes () and relative amplitude (a1~a3) of three components in the fitting data for the fluorescence lifetimes of fluorescent NDs (HeNDs), AuNDs, and HeNDs subjected to electron-beam irradiation (HeND_EB) for each experimental condition. τ1 (ns)

a1 (%)

τ2 (ns)

a2 (%)

τ3 (ns)

a3 (%)

(ns)

HeND (n=160)

0.51±0.14 62.1±12.7 4.90±2.63

19.2±7.5

34.21±13.0 18.7±11.7 28.6±12.9

Au-ND (n=173)

0.31±0.11 65.4±10.7 2.50±1.29

22.4±8.1

14.38±7.38

12.2±7.2

HeND_EB (n=148) 0.45±0.24 59.3±16.9 4.27±2.18

19.3±9.7

35.8±16.3

21.4±14.7 30.0±15.5

10.3±6.0

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Figure 2. Single-particle analysis of fluorescence lifetimes of Au-NDs and HeNDs. (a) Typical fluorescence decay profiles of Au-NDs (red; fitted data) and HeNDs (blue). (b) Distributions of values for Au-NDs (red) and HeNDs (blue).

To apply Au-NDs with the modulated optical properties as dualmodal cellular labeling probes in vitro, we injected Au-NDs into Hela cells. Twenty hours after the injection, we performed STEM-EDX analysis to investigate the stability and distribution of Au-NDs in a single cell. The viability tests implied that the administrated dose had no effect on the physiological functions (Figure S6). Figure 3(a) shows that the brighter spots in the HAADFSTEM images were found within small vesicle structures in a Hela cell. However, these spots could not be assigned only to Au-NDs because of the use of other heavy metal (Os, U, and Pb) signals to visualize the cytoplasmic proteins and cell organelles. To further confirm which bright spots originated from Au NPs, we performed element-selective EDX spectrum imaging. The EDX spectrum map clearly revealed that the bright spots in HAADF-STEM images correspond to Au NPs separated from HeNDs (C map) and background heavy metal signals (Os, U and Pb

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maps) that were derived from staining solutions (Figures 3(e)–(k), Figure S3(b), and S7). Comparison of the C map, Os map, U map, and Pb map demonstrated that the HeNDs were distinct from degraded or native biomolecules labelled with U, Os, or Pb. The element-selective images identified a single particle Au-ND with sub-nm scale spatial resolution. The results clearly indicated the physical and chemical stabilities of Au-NDs in physiological conditions, suggesting great potential for their use as surrogate markers in Hela cells.

Figure 3. Distribution of Au-NDs in a single mammalian cell. (a)–(d) HAADF-STEM images of a single Au-ND in a single vesicle of a Hela cell. NM: nuclear membrane, CM: cell membrane. (e) HAADF-STEM image of the region indicated by the red square in (d) and corresponding STEM-EDX elemental map ((f): Au/C map, (g) Au map, (h) C map, (i) Os map, (j) U map, and (k) Pb map). Scale bars: (a) 2 µm, (b) 500 nm, (c) 100 nm, (d) 20 nm, and (e)–(k) 5 nm.

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Finally, we evaluated the possibility of the visualization of Au-NDs as cell-labelling bioprobes by τf mapping. To confirm this idea, we injected HeNDs and Au-NDs into Hela cells as separate experiments. In conventional confocal microscopy experiments, the If map at wavelengths longer than 650 nm showed no difference between HeNDs and Au-NDs (Figures 4(a) and (c)). On the other hand, the map clearly demonstrated a difference in the distribution of τf and If for endocytosed Au-NDs in FLIM experiments (Figures 4(b) and (d)). In the τf map, fluorescence spectrum analysis showed that the regions of the bright spots contained NV centers. In TEM experiments, the many bright spots were found in cellular vesicle components, probably lysosomes to be confirmed as bright spots in fluorescence microscopy and in previous in vitro studies of cellular distribution analysis of HeNDs and Au-ND hybrid NPs.34 The of HeND was determined to be 15.4 ± 4.5 ns (n: numbers of particles analyzed; n = 11), whereas = 8.6 ± 2.3 ns (n = 15) for Au-NDs. The shortening effect of NV centers in AuNDs within cells on the suggests the idea of Au-NDs as dualmodal cell labeling probes, although cellular environments might affect the τf of HeNDs. In addition, Au-NDs were found to be highly stable against very acidic conditions in cells and no photobleaching behavior was observed (Figure S8). In general, NV centers show broad emission spectra, challenging the detection of multiple ND-labeled proteins or cells simultaneously. Based on the results obtained to date, the τf mapping protocol has the potential to simultaneously and continuously track the localization and motion properties of multiple molecules. In general, the τf of fluorescent probes (organic dyes and quantum dots etc.) is influenced by the cellular environment.40,41 Overall, the preference of the τf of Au-NDs being stable even in different cellular environments is suitable for tracking biomolecules, drugs, or cells of interest in vivo for a long time. In addition, Fig. 4 demonstrates that the FLIM mapping protocol based on values can clearly distinguish

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between the HeND-loaded cells and the Au-ND-loaded cells, even though the distribution of τf in the HeND and Au-NDs overlapped partially.40,41 Recently, separation and trapping techniques of NPs including fNDs using micro/nanofluidic devices have been developed.51-54 The establishment of the high-throughput separation protocols based on τf by FLIM could accelerate the sorting of HeNDs and Au-NDs to obtain a narrow distribution of τf values for each sample. If this is the case, the population of HeNDs or Au-NDs can be sorted into several groups with narrow distributions of τf. Hence, Au NPs can be attached on HeNDs with different τf distributions. Therefore, we can expect to simultaneously track more than three different cells or biomolecules labelled with Au-NDs having different τf distributions via τf-based dual-modal multi-color imaging in the future.

Figure 4. Comparison of the If map (a, c) and τf map (b, d) of the NV center of Au-NDs (a, b) and NDs (c, d) in a Hela cell. Scale bars: (a, c) 10 µm and (b, d) 2 µm.

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It is well known that Au NPs attached to biomaterials possessing thiol groups can be used as photothermal and photodynamic agents.55,56 Although increase in the diameter of AuNPs by further growth of Au-NDs might be required to induce these effects, photostable Au-NDs with specific targets like HER2-overexpressed receptors in ovarian cancer cells can be employed as theranostic probes in immunotherapy under optical microscopy.57-60 In addition, radiolysisinduced metal formation on NDs can lead to the formation of multiple types of metal NPs on NDs (Pt-NDs, Ag-NDs, and AgAu-NDs) with variable absorption spectra. The metal NPinduced plasmonic modulation of fluorescent NV centers and metal element-selective quantitative ratiometric imaging in STEM-EDX enable the multi-color labelling of multiple molecules or cells as dualmodal imaging probes in quantum sensing including environmental factors such as protein localization, temperature, and electronic potential. The physico-chemical stability and low toxicity under cellular conditions, and the possible multiple applications of these metal NPs and fluorescent ND hybrid nanoparticles as environmental biosensors and theranostic probes by surface modification, highlight the potential of dualmodal probes for longterm use in biomedical applications, as compared to other dualmodal imaging probes. Recently, several color centers with different fluorescence spectra have been reported for NDs, but the doping strategy for the formation of optical centers in small NDs (=

i =1 3

i

(3)

i =1

ai = Ampli . × 100 (%).

(4)

Atomic Force Microscopy (AFM) Measurements. AFM images were obtained using an MFP3D-BIO (Asylum Research, USA) mounted on the Olympus IX71 inverted fluorescence microscope. All the images were obtained using a tapping mode with silicon cantilevers (OMCLAC200TS, Olympus, Japan) at room temperature.

Preparation for Cell Culture Experiments. Hela cells were cultured in the following medium conditions: DMEM, 10% FBS, 1% penicillin/streptomycin in 95% air/5% CO2 at 37 °C. HeNDs and Au-NDs (100 µL of 1 mg/mL solutions) were mixed with 400 µL of the medium solution for 5 min at room temperature. Then an amount of medium equivalent to the injection volume was removed followed by the administration of mixed solutions of Au-NDs or NDs into to a culture medium. After 20 h, medium solutions were exchanged with a DMEM solution. FLIM experiments were performed in 95% air/5% CO2 at room temperature. The toxic effects of cells treated by both HeNDs and Au-NDs were evaluated by cell viability tests (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, USA). After 20 h, absorbance spectra (492 nm) were measured by a 96-plate reader (Multiskan FC, Thermo Scientific, USA). The statistical tests performed were two-sample t-tests (control and treatment).

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Preparation of Ultrathin Slices of Treated Hela Cells for TEM Experiments. Hela cells were cultured on a polystyrene cover slip, Cell Desk (Sumitomo Bakelite Co., Ltd., Japan), fixed with 2 % formaldehyde and 2.5 % glutaraldehyde in a 0.1 M sodium-phosphate buffer (pH 7.4) and washed three times for 5 min each time in the same buffer. The cells were post-fixed for 1 h with 1% osmium tetroxide and 1% potassium ferrocyanide in a 0.1 M sodium-phosphate buffer (pH 7.4), dehydrated in a graded ethanol series and embedded in Epon812 (TAAB Co. Ltd., UK). 80nm-thick ultra-thin sections were stained with saturated uranyl acetate and lead citrate solutions. Electron micrographs were obtained with a Cs-corrected transmission electron microscope (Titan, FEI Inc., USA) for STEM and EDX spectrum imaging. Quantitative analysis of the elemental composition in the sample was obtained by the Cliff-Lorimer method implemented on the ESPRIT software (Bruker Inc. Germany).

Supporting Information. A listing of the contents of each file supplied as Supporting Figures (Figures S1-S8). These materials are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]; [email protected]

Present Addresses. Masahito Morita, Office of Society-Academia Collaboration for Innovation, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

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Takashi Tachikawa, Molecular Photoscience Research Center, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan

ACKNOWLEDGMENTS This work has been partly supported by PRESTO, Japan Science and Technology Agency; the Cooperative Research Program of "Network Joint Research Center for Materials and Devices", Osaka University; the Innovative Project for Advanced Instruments, Renovation Center of Instruments for Science Education and Technology, Osaka University; and a Grant-inAid for Scientific Research (Projects 25220806, 23700536, and others) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government.

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