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High-Resolution Electron Microscopy and Spectroscopy of Ferritin in Biocompatible Graphene Liquid Cells and Graphene Sandwiches Canhui Wang, Qiao Qiao, Tolou Shokuhfar,* and Robert F. Klie* Cryo transmission electron microscopy (TEM) has been used extensively in modern biology and soft condensed matter physics research to study biological samples and bio-condensed matter interfaces. Since the vacuum environment in a TEM is incompatible with hydrated samples, cryo-samples are prepared by converting water into amorphous ice followed by sectioning;[1] however, these samples are no longer in their native state, and are most likely severely altered. In addition, radiation damage is a fundamentally limiting factor for spatial resolution when examining biological samples in a TEM. Even at liquid helium temperature, as used in cryo-TEM, the characterization of beam-sensitive material is limited by the beam-induced radiation damage, further altering the chemical and physical structure of the sample by local heating, electrostatic charging, or radiolysis.[2] However, there is a pressing need for characterization of biological samples in a liquid environment with atomic, or at least nm-scale resolution, to study their structures and dynamics. Here we show a novel approach of encapsulating biological samples using monolayers of graphene, thereby not only allowing biological samples to be directly imaged at high resolution in their native liquid state, but also enabling nm-scale analysis using electron energy-loss spectroscopy (EELS) to quantify the local atomic and electronic structures of biomaterials. Using ferritin as a model sample, we characterize the atomic and electronic structures of the ferri-hydride core and find a reduction of iron-oxide from Fe3+ to Fe2+ when the protein is in the hydrated state. We further demonstrate the ability of graphene to reduce the effects of electron-beam induced damage, which will enable
C. Wang, Dr. Q. Qiao,[+] Prof. R. F. Klie Department of Physics University of Illinois at Chicago Chicago, IL 60607, USA E-mail: [email protected] Prof. T. Shokuhfar Department of Mechanical Engineering-Engineering Mechanics MultiScale Technologies Institute Michigan Technological University Houghton, MI 49931, USA E-mail: [email protected] Prof. T. Shokuhfar Department of Physics and Department of Mechanical and Industrial Engineering University of Illinois at Chicago Chicago, IL 60607, USA [+] Present Address: Vanderbilt University Dept. of Physics & Astronomy Box 1807-B 6631 Stevenson Center Nashville, TN 37235, USA
DOI: 10.1002/adma.201306069 3410
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atomic-resolution imaging and nm-resolution spectroscopy of beam-sensitive materials. This approach is not limited to liquid samples and is used to characterize beam-sensitive dry materials sandwiched between two layers of graphene. To study liquid sample, several liquid cell designs that use electron transparent Si3N4 membranes have become commercially available in recent years that enable materials to be imaged in a carefully controlled liquid environment within a TEM. However, all suffer from a few key limitations that do not allow for atomic-resolution imaging or spectroscopy: 1) two thick Si3N4 layers (50–500 nm) are used as windows to separate the liquid from the TEM vacuum and 2) the thickness of the liquid surrounding the sample is usually between 50 nm and several microns.[3] Furthermore, in-situ chemical identification of materials in a liquid environment is also currently limited using such stages. While some X-ray analysis is possible with specially designed stages, EELS is degraded by multiple scattering events in the thick window layers, and the strong coreloss signals associated with the presence of Si and N.[4] In addition to the increase sample thickness, radiation damage is a fundamentally limiting factor when examining biological samples in TEM, limiting the spatial resolution to nanometers for direct imaging, and tens of nanometers for EELS[5] or energy-dispersive X-ray spectroscopy (EDX).[6] It has been shown that coating both sides of the specimen (with several nm thick of carbon or metal),[7–10] or lowering the temperature of the specimen to liquid N or He temperatures[7] have positive effects against radiation damage by reducing beam-induced temperature rise or electrostatic charging,[10] mass loss,[9] as well as the loss of crystallinity.[8] A recent study of MoS2 sandwiched between two layers of graphene has reported a reduction in defect formation rate.[11] These studies suggest that radiation damage in TEM can be controlled to limit the breakage of covalent bonds of the sample. The general graphene sandwich approach is, however, currently incompatible with biological samples due to the use of bio-incompatible substances such as acetone[11] or isopropanol (IPA)[12] during sample preparation. Moreover, further reduction of radiation damage is needed for characterization of biological samples, since many biological structures and functions are related to much weaker hydrogen bonds. In 2012, Yuk et al. reported that the formation of Pt crystals suspended in a liquid can be imaged with atomic resolution through encapsulating the liquid by monolayers of graphene.[13] It was further demonstrated that double strand DNA (dsDNA) connected to Au nanocrystals can be imaged at nanometer resolution in these graphene liquid cells (GLCs).[14] However, direct imaging or spectroscopy of soft materials utilizing this approach is still lacking, leaving applications of this new
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marginally on the size of the GLCs. (see Supporting Information and video 3). Although the electron dose rate of the probe commonly used for high-resolution analysis in scanning transmission electron microscopy (STEM) mode is 7 orders of magnitude higher than the above measured threshold dose rate, our experiments show that it is possible to avoid generating bubbles in STEM mode by keeping the area-averaged dose rate (which is proportional to magnification) below a threshold value of around 9 e− Å−2 s−1 (discussed in Supporting Information, also see video 3). These results suggest that below the threshold dose rate, the energy deposited by the incoming electrons is dissipated by the graphene cell system from the area irradiated with electron at a rate equivalent to the beam current of several electrons per Å2 per second. Further experiments show that at higher magnification (i.e. higher dose rate), the pixel dwell time can be decreased to avoid Figure 1. Schematic diagram (A), as well as STEM images (B and C) of ferritin molecules in GLCs and graphene sandwiches. B) An annular bright field (ABF) STEM image showing ferritin the formation of bubbles. This suggests that when using a sub-nm electron probes, the molecules encapsulated in both a GLC and graphene layers. The edges of the GLC is indicated by dashed lines. C) An HAADF STEM image showing atomic resolution image of a sandwiched pixel dwell time is an important parameter ferritin molecule, with the 12 nm in diameter protein shell. Inset: FFT from atomic resolution in determining the beam damage effects. ABF STEM image of the selected area showing patterns of two single graphene layers labeled Since it appears that the electron does rate g1 and g2, and iron core of ferritin labeled F. and pixel dwell time plays dominant roles in determining the radiation damage in GLC’s, as opposed to total electron dose, it now seems possible that generation of liquid cells for biochemical and biological probbeam sensitive samples can be imaged at atomic resolution by lems still unexplored.[15] accumulating signals using low electron dose rates and pixel We introduce a method that directly encapsulates biological dwell times. samples with or without liquid between two layers of freeUsing ferritin as a model system, we next show that direct standing graphene while avoiding bio-incompatible substances atomic-resolution imaging and chemical identification can be to remove or detach graphene from its support during sample achieved in biological molecules embedded in a thin liquid preparation (See Supporting Information and Figure S1, S2 within a GLC. Ferritin is a protein, 12 nm in diameter, formed and S3). We further show that using low dose rate imaging from a spherical protein shell (apoferritin) that surrounds a technique, the electron beam radiation damage can be reduced 6 nm in diameter core of hydrous ferric oxide. The biological to hydrogen bond breakage level. A schematic diagram of the function of ferritin includes storage and release of iron, which prepared sample is shown in Figure 1A. Using this sample will be affected by both the apoferritin structure and the liquid preparation method, ferritin from horse spleen is characterized environment. Stored in the form of Fe3+ ions, when released in the aberration-corrected JEOL ARM200CF STEM/TEM operated at a primary electron energy of 80 keV. The prepared GLCs from ferritin, the iron will first be reduced to Fe2+, and is then contain numerous areas of ferritin molecules in solution (Video readily lost from the protein molecule to its surrounding. 1), as well as areas where the water has drained leaving behind Figure 2 shows high-angle annular dark field (HAADF) and ferritin molecules sandwiched between two layers of graphene. annular bright field (ABF) STEM images of ferritin within a Figure 1B shows one such area containing both encapsulated liquid cell environment. Figure 2C shows lattice fringes of the liquid cells and sandwiched ferritin molecules. The presence of ferritin iron core in a liquid environment and Figure 2E shows a liquid is confirmed by increasing the electron dose rate and individual iron atoms in a water at the edge of a GLC. These generating gas bubbles inside the GLCs (Videos 1–3). The presatoms are also present in high concentrations near the ferritin ence of graphene encapsulating the sample is also confirmed iron core in an area without liquid (Figure 1C and Figure S5B). by atomic resolution imaging as shown in Figure 1C. Next, we examine the effects of the graphene cell on the While the formation of gas bubbles in a liquid cell during beam induced mass loss of the sample. Usually, a dose of electron irradiation is generally undesired, since it suggests 10 e− Å−2 will result in a mass loss of 20–80% of a biological physical and chemical changes of the system induced by the sample at room temperature. To examine the ability of graelectron beam,[16] we use the formation of bubbles to determine phene to reduce beam induced mass loss, ferritin sandwiched between two layers of graphene, as shown in Figure 3A, is the radiation damage critical dose rate threshold for a static examined using EEL spectrum imaging with a total dose of electron beam and find that the threshold dose rate for the 106 e− Å−2. At this dose, significant mass loss should occur in an formation of bubbles is around 6 e− Å−2 s−1 and depends only
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resolved with 1 nm resolution. In the case of graphene sandwiched sample, the shell contains nitrogen (N) and oxygen (O) where the protein shell should be, while in the case of unprotected sample, the O signal is mostly absent, suggesting significant loss of O due to beam irradiation. The graphene covering both surfaces of the sample in Figure 3A has prevented such mass loss even at high electron dose and high electron dose rate. This demonstrates that high resolution element mapping of beam sensitive materials will be possible using the biocompatible graphene sandwich approach. However, the EELS fine structure as shown in spectra 1 and 2 in Figure 4B clearly demonstrates that the local atomic structure of the protein is destroyed during these measurements. One of the most exciting aspects of the GLC design is that it allows for spectroscopic characterization of the samples using EELS, without the presence of Si or N absorption edges covering the edges of interest. Since long exposure under a focused electron beam Figure 2. ABF (A, B and C) and HAADF (D and E) STEM images of ferritin in GLCs. Bubbles in will cause radiation damage for beam sensi(A), (B) and (D) were formed in advance in Ronchigram mode to confirm the presence of liquid. tive samples even for graphene protected In STEM mode, electron dose rate and pixel dwell time is optimized so that no further bubbles samples (observed as a loss in fine structure are formed during scanning. FFT of a selected area containing a ferritin is shown as an inset in of the EEL spectra), a different approach (C). Lattice fringes of the ferritin iron core are resolved in (C) with a spacing measured to be 2.7 Å, consistent with the ferrihydrite in the (0–10) orientation. Single iron atoms are resolved is needed here. Our study of the radiation in a liquid environment in image (E) near the edge of a GLC. A line profile across the upper damage effects has shown that it is possible left atom is shown as an inset of (E), with each pixel corresponds to 0.99 Å. The resolution of to lower the radiation damage level in the these images is optimized by taking images under the corresponding threshold area averaged TEM by reducing the area averaged dose rate dose rate of bubble formation at each magnification. and pixel dwell time. Here we select a small area of interest form the GLC sample to be repeatedly scanned using a low pixel dwell time to accumulate unprotected sample. As a reference, Figure 3B shows a sample enough signal for core-loss EELS. This approach avoids gensitting only on a monolayer of graphene examined under the eration of bubbles or bursting the GLC, and limits the beam same conditions as a sample sandwiched between two layers of damage effects within or below hydrogen bond breakage. The graphene. The raw EELS data is filtered using Multivariate Staspatially averaged core-loss EEL spectra can then be obtained, tistical Analysis (MSA)[17] to obtain a better signal to noise ratio. but the spatial resolution of the spectra will be limited to the In both cases, a 12 nm in diameter shell of ferritin is clearly size of scanned area. By minimizing the pixel dwell time (∼1 µs), flyback time (250 µs), as well as the impinging probe current, an area as small as (1.3 nm)2 can be characterized without destroying the GLC or forming bubbles. Figure 4B shows core-loss EELS spectra of such areas at the center (spectrum 4), as well as at the edge of a ferritin molecule (spectrum 5) located inside the liquid. After acquisition of the EEL spectrum, the GLC is examined in STEM mode to confirm its integrity. The fine-structure of the N K- and O K-edges taken form the shell of ferritin in a liquid environment (spectrum 5) suggests Figure 3. EELS maps of ferritin molecules sandwiched between graphene sheets (A), and on a the presence of the protein. Spectrum 4 in monolayer graphene (B) with 1 nm resolution. The dose rate of the probe is 108 e− s−1. The dwell Figure 4B (taken at the center of the iron 2 8 − time per pixel is 1s. The electron dose of each pixel (1 nm ) is 10 e . The raw data is filtered using Multivariate Statistical Analysis (MSA)[17] to obtain a better signal to noise ratio. The protein core) confirms the presence of Fe, N, as well shell of ferritin is clearly resolved in both cases. The iron valence in the iron core is identified as as O. We attribute the Fe signal to the iron Fe3+ distributing across the whole core, as discussed in Supporting Information and Figure S6. core of ferritin, N signal to the protein shell
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ferritin molecule and the integrity and biological function of apoferritin, the protein shell of the ferritin, is preserved. This indicates the radiation damage is reduced below hydrogen bond breakage level due to the protection of graphene and the low dose rate technique, as discussed in Supporting Information. Atomic resolution imaging of relevant biochemical activity, the release and uptake of Fe atoms through the channels of ferritin protein shell, is shown in Figure S5 and discussed in Supporting Information. In summary, we have applied the concept of graphene liquid cells to general biological samples and examined in their native state using aberration-corrected STEM analysis. It Figure 4. HAADF STEM image of a GLC (A) and EELS spectra (B) of ferritin (1, 2, 4, 5) and was shown that graphene provides a radiawater (3). The bubble located at the center of the GLC is generated in advance to confirm exist- tion damage resistant mechanism allowing ence of a liquid in Ronchigram mode. EELS spectrum (1) is taken from the iron core of the for significantly increased spatial resoluEELS map shown in Figure 3A, (2) is taken from the protein shell of the EELS map shown in tion of beam sensitive sample using both Figure 3A, (3) is taken from water inside a GLC from a separate sample, (4) (5) were acquired imaging and spectroscopy. Our results dem2 through repeat scanning of a (1.3 nm) area located at the position shown in (A) with a below onstrate that, as opposed to conventional bubble formation threshold area averaged dose rate and a total scanning time of 1s. (4) is taken biological electron microscopy, such as cryo at the center of the iron core. (5) is taken at the edge of a ferritin molecule. electron microscopy, the electron dose rate, instead of the total electron dose, determines that is covering the iron core, and the O signal to a combinathe radiation damaging process in graphene wrapped samtion of liquid water, ferrihydrite core, and protein shell. For ples. We speculate that atomic-resolution three dimensional reference, spectrum 3 shows the EEL spectra taken from water structure of biological samples along with chemical bonding and spectra 1 and 2 are taken from graphene-sandwiched ferinformation can potentially be reconstructed by combining ritin shown in Figure 3A. The near-edge fine structure of N is this method with electron tomography or single-particle resolved in spectra 4 and 5, while it is not visible in spectra 1 microscopy. and 2. The difference between those spectra (other than the presence of water) is that in spectra 1 and 2, the signals are acquired using a stationary probe, while spectra 4 and 5 are Experimental Section acquired while scanning the probe with an area averaged dose rate below damage threshold. The N K-edge fine-structure in Purified ferritin molecules (Sigma Aldrich, Prod. # F4503, diluted 4 and 5 is similar to the structure of EEL spectrum reported 500 times with deionized water) is characterized in the aberrationfor CN crystal,[18] suggesting bonding structure between N and corrected JEOL JEM-ARM200CF operated at a primary electron energy of 80 keV. The microscope is equipped with a cold field-emission either H, O, or C. This result shows that due to the protective source, which yields an energy resolution of 0.35 eV and 1.2 Å spatial effect of graphene, the fine structure of the EEL spectrum (and resolution with the probe spherical-aberration corrector. The probe thereby the local atomic and electronic structures) can be precurrent of the microscope is calibrated using a faraday cup. Videos in served in beam sensitive materials by using a low dose rate Ronchigram mode are taken with a 0.033 s per frame exposure time. accumulation of electron. All videos plays in real time. STEM images are collected simultaneously Finally, careful quantification of the Fe L-edges (see Supon bright field and annular dark field detectors with dwell times ranging from 1 µs to 150 µs per pixel. With an emission current of 15 µA, the porting Information and Figure S6) allows us to determine STEM probe had a current density of 4.3 pA, corresponding to aperture the local Fe oxidation states. The valence states of Fe can be size of 20 µm, and estimated 272 pA corresponding to aperture size of quantified using the Fe L3 and L2 peaks, including their widths 150 µm. A convergence semi-angle of 22 mrad (for 40 µm aperture) and and relative positions.[19,20] We find a chemical shift of the Fe L3 11mrad (for 20 µm aperture) is used for both STEM imaging and EELS. peak to higher energy by 2 eV and an increased width of the L3 For HAADF STEM imaging, a 90 mrad collection inner angle is used; peaks (Figure S6), suggesting that Fe is in the 3+ valence state, for ABF imaging, 11–22 mrad collection semi-angle is used. For EELS, for ferritin in the absence of water. Fe3+ is the thermodynamia collection semi-angle of 71 mrad is used. Digital Micrograph (Gatan, Inc., USA) is utilized for all data acquisition. cally stable valence state of iron in ferritin, while in the case of the hydrated ferritin located inside a GLC, a mixture of Fe2+ and Fe3+ is present. We estimate the concentration of Fe2+ to be 81% by fitting the signal using the method described in reference.[19] Supporting Information This reduction of iron valence to 2+ state in the hydrated ferritin suggests that in the liquid environment the initial stages of iron Supporting Information is available from the Wiley Online Library or from the author. release by ferritin[21] are measured at nm resolution in a single
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Acknowledgements The authors would like to acknowledge the Research Resource Center of UIC for providing instrumentation support, Zhejiang University Dr. Hongtao Wang’s research group and Dr. Anmin Nie for providing the CVD graphene, Drs. W. Hendrickson, A.W. Nicholls and K.B. Low for the helpful discussions. This work is funded by Michigan Technological University. The UIC JEOL JEM-ARM200CF is supported by an MRI-R2 grant from the National Science Foundation (Grant No. DMR-0959470). C.W. acquired and processed the data, C.W. and Q.Q. acquired and processed the EELS data, C.W., Q.Q., T.S. and R.F.K. discussed the results and drafted the manuscript. Received: December 11, 2013 Revised: January 13, 2014 Published online: February 3, 2014
[1] a) H. Stahlberg, T. Walz, ACS Chem. Biol. 2008, 3, 268; b) J. Pierson, M. Sani, C. Tomova, S. Godsave, P. J. Peters, Histochem. Cell Biol. 2009, 132, 253. [2] R. F. Egerton, P. Li, M. Malac, Micron 2004, 35, 399. [3] N. de Jonge, F. M. Ross, Nat. Nanotechnol. 2011, 6, 695. [4] M. E. Holtz, Y. Yu, J. Gao, H. D. Abruña, D. A. Muller, Microsc. Microanal. 2012, 18, 1094. [5] R. F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, Springer, 2011.
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[6] J. Wu, A. Kim, R. Bleher, B. Myers, R. Marvin, H. Inada, K. Nakamura, X. Zhang, E. Roth, S. Li, Ultramicroscopy 2013. [7] R. Egerton, P. Crozier, P. Rice, Ultramicroscopy 1987, 23, 305. [8] J. Fryer, F. Holland, Proc. R. Soc. London A – Math. Phys. Sci 1984, 393, 353. [9] J. Fryer, F. Holland, Ultramicroscopy 1983, 11, 67. [10] S. Salih, V. Cosslett, Phil. Mag. 1974, 30, 225. [11] R. Zan, Q. M. Ramasse, R. Jalil, T. Georgiou, U. Bangert, K. S. Novoselov, ACS Nano 2013. [12] J. M. Yuk, K. Kim, B. Aleman, W. Regan, J. H. Ryu, J. Park, P. Ercius, H. M. Lee, A. P. Alivisatos, M. F. Crommie, J. Y. Lee, A. Zettl, Nano Lett. 2011, 11, 3290. [13] J. M. Yuk, J. Park, P. Ercius, K. Kim, D. J. Hellebusch, M. F. Crommie, J. Y. Lee, A. Zettl, A. P. Alivisatos, Science 2012, 336, 61. [14] Q. Chen, J. M. Smith, J. Park, K. Kim, D. Ho, H. I. Rasool, A. Zettl, A. P. Alivisatos, Nano Lett. 2013, 13, 4556. [15] C. Colliex, Science 2012, 336, 44. [16] B. C. Garrett, D. A. Dixon, D. M. Camaioni, D. M. Chipman, M. A. Johnson, C. D. Jonah, G. A. Kimmel, J. H. Miller, T. N. Rescigno, P. J. Rossky, Chem. Rev. 2005, 105, 355. [17] N. Borglund, P.-G. Åstrand, S. Csillag, Microsc. Microanal. 2005, 11, 88. [18] S. Trasobares, S. Gao, O. Stéphan, A. Gloter, C. Colliex, J. Zhu, Chem. Phys. Lett. 2002, 352, 12. [19] L. A. Garvie, P. R. Buseck, Nature 1998, 396, 667. [20] C. Colliex, T. Manoubi, C. Ortiz, Phys. Rev. B 1991, 44, 11402. [21] G. Watt, R. B. Frankel, G. Papaefthymiou, Proc. Nati. Acad. Sci. USA 1985, 82, 3640.
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High-Resolution Electron Microscopy and Spectroscopy of Ferritin in Biocompatible Graphene Liquid Cells and Graphene Sandwiches Canhui Wang, Qiao Qiao, Tolou Shokuhfar,* and Robert F. Klie* Cryo transmission electron microscopy (TEM) has been used extensively in modern biology and soft condensed matter physics research to study biological samples and bio-condensed matter interfaces. Since the vacuum environment in a TEM is incompatible with hydrated samples, cryo-samples are prepared by converting water into amorphous ice followed by sectioning;[1] however, these samples are no longer in their native state, and are most likely severely altered. In addition, radiation damage is a fundamentally limiting factor for spatial resolution when examining biological samples in a TEM. Even at liquid helium temperature, as used in cryo-TEM, the characterization of beam-sensitive material is limited by the beam-induced radiation damage, further altering the chemical and physical structure of the sample by local heating, electrostatic charging, or radiolysis.[2] However, there is a pressing need for characterization of biological samples in a liquid environment with atomic, or at least nm-scale resolution, to study their structures and dynamics. Here we show a novel approach of encapsulating biological samples using monolayers of graphene, thereby not only allowing biological samples to be directly imaged at high resolution in their native liquid state, but also enabling nm-scale analysis using electron energy-loss spectroscopy (EELS) to quantify the local atomic and electronic structures of biomaterials. Using ferritin as a model sample, we characterize the atomic and electronic structures of the ferri-hydride core and find a reduction of iron-oxide from Fe3+ to Fe2+ when the protein is in the hydrated state. We further demonstrate the ability of graphene to reduce the effects of electron-beam induced damage, which will enable
C. Wang, Dr. Q. Qiao,[+] Prof. R. F. Klie Department of Physics University of Illinois at Chicago Chicago, IL 60607, USA E-mail: [email protected] Prof. T. Shokuhfar Department of Mechanical Engineering-Engineering Mechanics MultiScale Technologies Institute Michigan Technological University Houghton, MI 49931, USA E-mail: [email protected] Prof. T. Shokuhfar Department of Physics and Department of Mechanical and Industrial Engineering University of Illinois at Chicago Chicago, IL 60607, USA [+] Present Address: Vanderbilt University Dept. of Physics & Astronomy Box 1807-B 6631 Stevenson Center Nashville, TN 37235, USA
DOI: 10.1002/adma.201306069 3410
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atomic-resolution imaging and nm-resolution spectroscopy of beam-sensitive materials. This approach is not limited to liquid samples and is used to characterize beam-sensitive dry materials sandwiched between two layers of graphene. To study liquid sample, several liquid cell designs that use electron transparent Si3N4 membranes have become commercially available in recent years that enable materials to be imaged in a carefully controlled liquid environment within a TEM. However, all suffer from a few key limitations that do not allow for atomic-resolution imaging or spectroscopy: 1) two thick Si3N4 layers (50–500 nm) are used as windows to separate the liquid from the TEM vacuum and 2) the thickness of the liquid surrounding the sample is usually between 50 nm and several microns.[3] Furthermore, in-situ chemical identification of materials in a liquid environment is also currently limited using such stages. While some X-ray analysis is possible with specially designed stages, EELS is degraded by multiple scattering events in the thick window layers, and the strong coreloss signals associated with the presence of Si and N.[4] In addition to the increase sample thickness, radiation damage is a fundamentally limiting factor when examining biological samples in TEM, limiting the spatial resolution to nanometers for direct imaging, and tens of nanometers for EELS[5] or energy-dispersive X-ray spectroscopy (EDX).[6] It has been shown that coating both sides of the specimen (with several nm thick of carbon or metal),[7–10] or lowering the temperature of the specimen to liquid N or He temperatures[7] have positive effects against radiation damage by reducing beam-induced temperature rise or electrostatic charging,[10] mass loss,[9] as well as the loss of crystallinity.[8] A recent study of MoS2 sandwiched between two layers of graphene has reported a reduction in defect formation rate.[11] These studies suggest that radiation damage in TEM can be controlled to limit the breakage of covalent bonds of the sample. The general graphene sandwich approach is, however, currently incompatible with biological samples due to the use of bio-incompatible substances such as acetone[11] or isopropanol (IPA)[12] during sample preparation. Moreover, further reduction of radiation damage is needed for characterization of biological samples, since many biological structures and functions are related to much weaker hydrogen bonds. In 2012, Yuk et al. reported that the formation of Pt crystals suspended in a liquid can be imaged with atomic resolution through encapsulating the liquid by monolayers of graphene.[13] It was further demonstrated that double strand DNA (dsDNA) connected to Au nanocrystals can be imaged at nanometer resolution in these graphene liquid cells (GLCs).[14] However, direct imaging or spectroscopy of soft materials utilizing this approach is still lacking, leaving applications of this new
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marginally on the size of the GLCs. (see Supporting Information and video 3). Although the electron dose rate of the probe commonly used for high-resolution analysis in scanning transmission electron microscopy (STEM) mode is 7 orders of magnitude higher than the above measured threshold dose rate, our experiments show that it is possible to avoid generating bubbles in STEM mode by keeping the area-averaged dose rate (which is proportional to magnification) below a threshold value of around 9 e− Å−2 s−1 (discussed in Supporting Information, also see video 3). These results suggest that below the threshold dose rate, the energy deposited by the incoming electrons is dissipated by the graphene cell system from the area irradiated with electron at a rate equivalent to the beam current of several electrons per Å2 per second. Further experiments show that at higher magnification (i.e. higher dose rate), the pixel dwell time can be decreased to avoid Figure 1. Schematic diagram (A), as well as STEM images (B and C) of ferritin molecules in GLCs and graphene sandwiches. B) An annular bright field (ABF) STEM image showing ferritin the formation of bubbles. This suggests that when using a sub-nm electron probes, the molecules encapsulated in both a GLC and graphene layers. The edges of the GLC is indicated by dashed lines. C) An HAADF STEM image showing atomic resolution image of a sandwiched pixel dwell time is an important parameter ferritin molecule, with the 12 nm in diameter protein shell. Inset: FFT from atomic resolution in determining the beam damage effects. ABF STEM image of the selected area showing patterns of two single graphene layers labeled Since it appears that the electron does rate g1 and g2, and iron core of ferritin labeled F. and pixel dwell time plays dominant roles in determining the radiation damage in GLC’s, as opposed to total electron dose, it now seems possible that generation of liquid cells for biochemical and biological probbeam sensitive samples can be imaged at atomic resolution by lems still unexplored.[15] accumulating signals using low electron dose rates and pixel We introduce a method that directly encapsulates biological dwell times. samples with or without liquid between two layers of freeUsing ferritin as a model system, we next show that direct standing graphene while avoiding bio-incompatible substances atomic-resolution imaging and chemical identification can be to remove or detach graphene from its support during sample achieved in biological molecules embedded in a thin liquid preparation (See Supporting Information and Figure S1, S2 within a GLC. Ferritin is a protein, 12 nm in diameter, formed and S3). We further show that using low dose rate imaging from a spherical protein shell (apoferritin) that surrounds a technique, the electron beam radiation damage can be reduced 6 nm in diameter core of hydrous ferric oxide. The biological to hydrogen bond breakage level. A schematic diagram of the function of ferritin includes storage and release of iron, which prepared sample is shown in Figure 1A. Using this sample will be affected by both the apoferritin structure and the liquid preparation method, ferritin from horse spleen is characterized environment. Stored in the form of Fe3+ ions, when released in the aberration-corrected JEOL ARM200CF STEM/TEM operated at a primary electron energy of 80 keV. The prepared GLCs from ferritin, the iron will first be reduced to Fe2+, and is then contain numerous areas of ferritin molecules in solution (Video readily lost from the protein molecule to its surrounding. 1), as well as areas where the water has drained leaving behind Figure 2 shows high-angle annular dark field (HAADF) and ferritin molecules sandwiched between two layers of graphene. annular bright field (ABF) STEM images of ferritin within a Figure 1B shows one such area containing both encapsulated liquid cell environment. Figure 2C shows lattice fringes of the liquid cells and sandwiched ferritin molecules. The presence of ferritin iron core in a liquid environment and Figure 2E shows a liquid is confirmed by increasing the electron dose rate and individual iron atoms in a water at the edge of a GLC. These generating gas bubbles inside the GLCs (Videos 1–3). The presatoms are also present in high concentrations near the ferritin ence of graphene encapsulating the sample is also confirmed iron core in an area without liquid (Figure 1C and Figure S5B). by atomic resolution imaging as shown in Figure 1C. Next, we examine the effects of the graphene cell on the While the formation of gas bubbles in a liquid cell during beam induced mass loss of the sample. Usually, a dose of electron irradiation is generally undesired, since it suggests 10 e− Å−2 will result in a mass loss of 20–80% of a biological physical and chemical changes of the system induced by the sample at room temperature. To examine the ability of graelectron beam,[16] we use the formation of bubbles to determine phene to reduce beam induced mass loss, ferritin sandwiched between two layers of graphene, as shown in Figure 3A, is the radiation damage critical dose rate threshold for a static examined using EEL spectrum imaging with a total dose of electron beam and find that the threshold dose rate for the 106 e− Å−2. At this dose, significant mass loss should occur in an formation of bubbles is around 6 e− Å−2 s−1 and depends only
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resolved with 1 nm resolution. In the case of graphene sandwiched sample, the shell contains nitrogen (N) and oxygen (O) where the protein shell should be, while in the case of unprotected sample, the O signal is mostly absent, suggesting significant loss of O due to beam irradiation. The graphene covering both surfaces of the sample in Figure 3A has prevented such mass loss even at high electron dose and high electron dose rate. This demonstrates that high resolution element mapping of beam sensitive materials will be possible using the biocompatible graphene sandwich approach. However, the EELS fine structure as shown in spectra 1 and 2 in Figure 4B clearly demonstrates that the local atomic structure of the protein is destroyed during these measurements. One of the most exciting aspects of the GLC design is that it allows for spectroscopic characterization of the samples using EELS, without the presence of Si or N absorption edges covering the edges of interest. Since long exposure under a focused electron beam Figure 2. ABF (A, B and C) and HAADF (D and E) STEM images of ferritin in GLCs. Bubbles in will cause radiation damage for beam sensi(A), (B) and (D) were formed in advance in Ronchigram mode to confirm the presence of liquid. tive samples even for graphene protected In STEM mode, electron dose rate and pixel dwell time is optimized so that no further bubbles samples (observed as a loss in fine structure are formed during scanning. FFT of a selected area containing a ferritin is shown as an inset in of the EEL spectra), a different approach (C). Lattice fringes of the ferritin iron core are resolved in (C) with a spacing measured to be 2.7 Å, consistent with the ferrihydrite in the (0–10) orientation. Single iron atoms are resolved is needed here. Our study of the radiation in a liquid environment in image (E) near the edge of a GLC. A line profile across the upper damage effects has shown that it is possible left atom is shown as an inset of (E), with each pixel corresponds to 0.99 Å. The resolution of to lower the radiation damage level in the these images is optimized by taking images under the corresponding threshold area averaged TEM by reducing the area averaged dose rate dose rate of bubble formation at each magnification. and pixel dwell time. Here we select a small area of interest form the GLC sample to be repeatedly scanned using a low pixel dwell time to accumulate unprotected sample. As a reference, Figure 3B shows a sample enough signal for core-loss EELS. This approach avoids gensitting only on a monolayer of graphene examined under the eration of bubbles or bursting the GLC, and limits the beam same conditions as a sample sandwiched between two layers of damage effects within or below hydrogen bond breakage. The graphene. The raw EELS data is filtered using Multivariate Staspatially averaged core-loss EEL spectra can then be obtained, tistical Analysis (MSA)[17] to obtain a better signal to noise ratio. but the spatial resolution of the spectra will be limited to the In both cases, a 12 nm in diameter shell of ferritin is clearly size of scanned area. By minimizing the pixel dwell time (∼1 µs), flyback time (250 µs), as well as the impinging probe current, an area as small as (1.3 nm)2 can be characterized without destroying the GLC or forming bubbles. Figure 4B shows core-loss EELS spectra of such areas at the center (spectrum 4), as well as at the edge of a ferritin molecule (spectrum 5) located inside the liquid. After acquisition of the EEL spectrum, the GLC is examined in STEM mode to confirm its integrity. The fine-structure of the N K- and O K-edges taken form the shell of ferritin in a liquid environment (spectrum 5) suggests Figure 3. EELS maps of ferritin molecules sandwiched between graphene sheets (A), and on a the presence of the protein. Spectrum 4 in monolayer graphene (B) with 1 nm resolution. The dose rate of the probe is 108 e− s−1. The dwell Figure 4B (taken at the center of the iron 2 8 − time per pixel is 1s. The electron dose of each pixel (1 nm ) is 10 e . The raw data is filtered using Multivariate Statistical Analysis (MSA)[17] to obtain a better signal to noise ratio. The protein core) confirms the presence of Fe, N, as well shell of ferritin is clearly resolved in both cases. The iron valence in the iron core is identified as as O. We attribute the Fe signal to the iron Fe3+ distributing across the whole core, as discussed in Supporting Information and Figure S6. core of ferritin, N signal to the protein shell
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ferritin molecule and the integrity and biological function of apoferritin, the protein shell of the ferritin, is preserved. This indicates the radiation damage is reduced below hydrogen bond breakage level due to the protection of graphene and the low dose rate technique, as discussed in Supporting Information. Atomic resolution imaging of relevant biochemical activity, the release and uptake of Fe atoms through the channels of ferritin protein shell, is shown in Figure S5 and discussed in Supporting Information. In summary, we have applied the concept of graphene liquid cells to general biological samples and examined in their native state using aberration-corrected STEM analysis. It Figure 4. HAADF STEM image of a GLC (A) and EELS spectra (B) of ferritin (1, 2, 4, 5) and was shown that graphene provides a radiawater (3). The bubble located at the center of the GLC is generated in advance to confirm exist- tion damage resistant mechanism allowing ence of a liquid in Ronchigram mode. EELS spectrum (1) is taken from the iron core of the for significantly increased spatial resoluEELS map shown in Figure 3A, (2) is taken from the protein shell of the EELS map shown in tion of beam sensitive sample using both Figure 3A, (3) is taken from water inside a GLC from a separate sample, (4) (5) were acquired imaging and spectroscopy. Our results dem2 through repeat scanning of a (1.3 nm) area located at the position shown in (A) with a below onstrate that, as opposed to conventional bubble formation threshold area averaged dose rate and a total scanning time of 1s. (4) is taken biological electron microscopy, such as cryo at the center of the iron core. (5) is taken at the edge of a ferritin molecule. electron microscopy, the electron dose rate, instead of the total electron dose, determines that is covering the iron core, and the O signal to a combinathe radiation damaging process in graphene wrapped samtion of liquid water, ferrihydrite core, and protein shell. For ples. We speculate that atomic-resolution three dimensional reference, spectrum 3 shows the EEL spectra taken from water structure of biological samples along with chemical bonding and spectra 1 and 2 are taken from graphene-sandwiched ferinformation can potentially be reconstructed by combining ritin shown in Figure 3A. The near-edge fine structure of N is this method with electron tomography or single-particle resolved in spectra 4 and 5, while it is not visible in spectra 1 microscopy. and 2. The difference between those spectra (other than the presence of water) is that in spectra 1 and 2, the signals are acquired using a stationary probe, while spectra 4 and 5 are Experimental Section acquired while scanning the probe with an area averaged dose rate below damage threshold. The N K-edge fine-structure in Purified ferritin molecules (Sigma Aldrich, Prod. # F4503, diluted 4 and 5 is similar to the structure of EEL spectrum reported 500 times with deionized water) is characterized in the aberrationfor CN crystal,[18] suggesting bonding structure between N and corrected JEOL JEM-ARM200CF operated at a primary electron energy of 80 keV. The microscope is equipped with a cold field-emission either H, O, or C. This result shows that due to the protective source, which yields an energy resolution of 0.35 eV and 1.2 Å spatial effect of graphene, the fine structure of the EEL spectrum (and resolution with the probe spherical-aberration corrector. The probe thereby the local atomic and electronic structures) can be precurrent of the microscope is calibrated using a faraday cup. Videos in served in beam sensitive materials by using a low dose rate Ronchigram mode are taken with a 0.033 s per frame exposure time. accumulation of electron. All videos plays in real time. STEM images are collected simultaneously Finally, careful quantification of the Fe L-edges (see Supon bright field and annular dark field detectors with dwell times ranging from 1 µs to 150 µs per pixel. With an emission current of 15 µA, the porting Information and Figure S6) allows us to determine STEM probe had a current density of 4.3 pA, corresponding to aperture the local Fe oxidation states. The valence states of Fe can be size of 20 µm, and estimated 272 pA corresponding to aperture size of quantified using the Fe L3 and L2 peaks, including their widths 150 µm. A convergence semi-angle of 22 mrad (for 40 µm aperture) and and relative positions.[19,20] We find a chemical shift of the Fe L3 11mrad (for 20 µm aperture) is used for both STEM imaging and EELS. peak to higher energy by 2 eV and an increased width of the L3 For HAADF STEM imaging, a 90 mrad collection inner angle is used; peaks (Figure S6), suggesting that Fe is in the 3+ valence state, for ABF imaging, 11–22 mrad collection semi-angle is used. For EELS, for ferritin in the absence of water. Fe3+ is the thermodynamia collection semi-angle of 71 mrad is used. Digital Micrograph (Gatan, Inc., USA) is utilized for all data acquisition. cally stable valence state of iron in ferritin, while in the case of the hydrated ferritin located inside a GLC, a mixture of Fe2+ and Fe3+ is present. We estimate the concentration of Fe2+ to be 81% by fitting the signal using the method described in reference.[19] Supporting Information This reduction of iron valence to 2+ state in the hydrated ferritin suggests that in the liquid environment the initial stages of iron Supporting Information is available from the Wiley Online Library or from the author. release by ferritin[21] are measured at nm resolution in a single
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Acknowledgements The authors would like to acknowledge the Research Resource Center of UIC for providing instrumentation support, Zhejiang University Dr. Hongtao Wang’s research group and Dr. Anmin Nie for providing the CVD graphene, Drs. W. Hendrickson, A.W. Nicholls and K.B. Low for the helpful discussions. This work is funded by Michigan Technological University. The UIC JEOL JEM-ARM200CF is supported by an MRI-R2 grant from the National Science Foundation (Grant No. DMR-0959470). C.W. acquired and processed the data, C.W. and Q.Q. acquired and processed the EELS data, C.W., Q.Q., T.S. and R.F.K. discussed the results and drafted the manuscript. Received: December 11, 2013 Revised: January 13, 2014 Published online: February 3, 2014
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