NIH Public Access Author Manuscript Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
NIH-PA Author Manuscript
Published in final edited form as: Mater Res Soc Symp Proc. 2013 May 13; 1569: 157–163.
Advanced Characterization Techniques for Nanoparticles for Cancer Research: Applications of SEM and NanoSIMS for Locating Au Nanoparticles in Cells Paul J Kempen1, Chuck Hitzman2, Laura S Sasportas3, Sanjiv S Gambhir3, and Robert Sinclair1 1Department
of Material Science and Engineering, Stanford University, 496 Lomita Mall, Stanford, CA 94305-4034 U.S.A.
2Stanford
Nanocharacterization Laboratory, Stanford University, 476 Lomita Mall, Stanford, CA 94305-4035 U.S.A. 3Molecular
NIH-PA Author Manuscript
Imaging Program at Stanford, Department of Radiology, Stanford University, 318 Campus Drive, Stanford, CA 94305-5427 U.S.A.
Abstract The ability of nano secondary ion mass spectrometry (NanoSIMS) to locate and analyze Raman active gold core nanoparticles (R-AuNPs) in a biological system is compared with the standard analysis using the scanning electron microscope (SEM). The same cell with R-AuNPs on and inside the macrophage was analyzed with both techniques to directly compare them. SEM analysis showed a large number of nanoparticles within the cell. Subsequent NanoSIMS analysis showed fewer R-AuNPs with lower spatial resolution. SEM was determined to be superior to NanoSIMS for the analysis of inorganic nanoparticles in complex biological systems.
INTRODUCTION
NIH-PA Author Manuscript
With the continuing and growing use of nanoparticles for biological applications [1–3] it is becoming increasingly important to be able to accurately locate and characterize them on and within a cell. Owing to their small size it is necessary to utilize advanced characterization techniques to study them. The scanning electron microscope (SEM), capable of sub-nanometer spatial resolution [4] and good depth of field, is commonly utilized for this purpose [5]. Moreover, it is possible to distinguish materials by atomic number through the use of backscattered electron (BSE) imaging [6]. This atomic number sensitivity creates contrast in the image where higher atomic number inorganic nanoparticles appear bright against a dark low atomic number, organic matrix background. This enables the ready identification of inorganic nanoparticles both on the surface and in the cytoplasm of cell samples [7]. Recently there have been advances in secondary ion mass spectrometry (SIMS), combining SIMS with a focused scanning ion beam, allowing for the creation of high spatial resolution, ca 50 nm, compositional maps [8]. The NanoSIMS combines reasonably good spatial resolution with high atomic sensitivity, down to the ppm regime [8] and is powerful because
Kempen et al.
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NIH-PA Author Manuscript
it is capable of distinguishing between isotopes of the same material while analyzing up to seven distinct elements or species at the same time [9]. This technique has been increasingly utilized to study and analyze complex biological systems including the use of isotope labeling [10, 11]. Gold-core silica-shell nanoparticles (R-AuNPs) are promising as a multimodality surface enhanced Raman spectroscopy (SERS) and photoacoustic nanoparticle for diagnostic purposes [12–14]. R-AuNPs, produced by Oxonica Materials Inc (now owned by Cabot Corporation), consist of a 60 nm diameter gold core surrounded by a 30 nm thick silica shell. A monolayer of Raman active dye is adsorbed onto the surface of the gold core providing a surface enhancement effect that can improve the Raman signal intensity by many orders of magnitude [15]. SEM is an established technique for analyzing nanoparticles and has previously been utilized to locate and analyze R-AuNPs in a number of biological systems including T-cells [16] and brain tumors [12]. In this work SEM analysis of RAW264.7 human macrophages incubated with R-AuNPs was compared with NanoSIMS analysis of the same area to determine the viability of NanoSIMS as a technique to locate and characterize inorganic nanoparticles in biological systems.
NIH-PA Author Manuscript
EXPERIMENT R-AuNPs were incubated with RAW264.7 human macrophages grown on glass cover slips to induce cellular uptake of the nanoparticles. The cells were then fixed in a solution of 2.5% glutaraldehyde 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 1 hour at room temperature. The samples were then dehydrated in increasing concentrations of ethanol; 50%, 70%, 95% and 100% twice, at 20 minute intervals. The cells were then prepared for SEM and NanoSIMS analysis through critical point drying (CPD), creating a vacuum compatible sample while preserving the structure of the cells [17]. The glass cover slips were then placed on aluminum SEM stubs and coated with a thin layer of Au-Pd to improve conduction. The samples were then cross hatched with a diamond scribe to allow for co-localization between the SEM and the NanoSIMS.
NIH-PA Author Manuscript
The samples were imaged in the SEM using a FEI Magellan 400 XHR SEM operated at 15 kV with a probe current of 50 pA. After imaging in the SEM, the location was recorded by acquiring a low magnification montage image of the cover slip using the automated software in the Magellan and the sample was placed in the Cameca NanoSIMS 50L. Cesium+ primary ion beam bombardment and negative secondary ion detection were used to optimize the sensitivity for Au. The primary beam current at the sample was on the order of 1pA with an impact energy of 16 keV normal to the surface. Using the secondary electron signal generated by the scanning cesium, Cs+, ion beam the exact location was located. Composition maps for C2−, CN−, O−, Si−, P− and Au− were then generated to show the location of the R-AuNPs within the cell.
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DISCUSSION NIH-PA Author Manuscript
Scanning Electron Microscopy
NIH-PA Author Manuscript
Scanning electron microscopy is a powerful technique for analyzing R-AuNPs on and inside cells. Secondary electron (SE) imaging provides excellent surface topography information, as shown in figure 1(a–b), while BSE imaging can provide elemental information about the sample as shown in figure 1(c–d). The surface structure is more identifiable in the SE images while the R-AuNPs are can clearly be identified on the surface of the macrophage, appearing as bright areas in the BSE image due to the high atomic number gold core. Figure 1 is representative of the types of images that can be obtained from the SEM. SEM analysis indicated that the R-AuNPs were located across the surface of the macrophages, making this a useful system for comparison with NanoSIMS. A BSE image of the cell that was analyzed using the NanoSIMS is shown in figure 2, with a higher magnification image of the area analyzed for R-AuNPs. This image shows a large number of nanoparticles in the area of interest on or in the cell. BSE imaging can image R-AuNPs that are not at the surface of the sample because the high energy electron beam can penetrate into the sample creating signal several hundred nanometers below the surface of the cell. Some of the R-AuNPs shown in figure 2(B) are located within the cell and not on the surface. This cell was chosen because of the large extension in the upper part region of the cell allowing for easier identification in the NanoSIMS. Nano Secondary Ion Mass Spectrometry
NIH-PA Author Manuscript
Locating the same cell in the NanoSIMS is a non-trivial endeavor due to differences in image formation and quality. There is an image inversion in the NanoSIMS that must be accounted for when navigating and locating a cell. Once this is taken into account, there are still large differences in image quality due to the ion beam. This beam has a much larger probe diameter, around 50 nm, than the electron beam in the SEM, approximately 1 nm, resulting in decreased surface resolution, shown in figure 3. Additionally, the ion beam quickly sputters the thin layer of Au-Pd on the surface of the sample, exposing the underlying cell and glass substrate leading to localized charging. The cell in figure 3(a), the same cell shown in figure 2, appears more blurred as a result of the decreased resolution. There are also bright regions in the image that do not correspond to R-AuNPs but are rather due to charging of the sample. This makes identification of the same region between the two instruments difficult. The unusual shape of this cell was the single identifiable feature between the two techniques. It took about four hours to locate this specific cell in the NanoSIMS. Once the cell was identified at low magnification, the region of the cell indicated in the white box in figure 3 was analyzed for the presence of C2−, CN−, O−, Si−, P− and Au−. Initially no gold was identified in this region. This happened because the R-AuNPs are coated with silica preventing the sputtering of the underlying gold and the nanoparticles were located within the cell in this region requiring the sputtering of the surface of the cell before the R-AuNPs would become visible. NanoSIMS is essentially a surface sensitive technique because ion generation and sputtering can only occur within the first 1–2 nm of the surface. After slowly sputtering the surface away for approximately 30 minutes to
Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
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expose the R-AuNPs, compositional maps for C2−, CN−, O−, Si−, P− and Au− were created, figure 4.
NIH-PA Author Manuscript
The composition maps show the location of the cell in the analyzed region as well as some of the R-AuNPs located in that region. The C2−, CN− and P− maps show the structure of the cell. Both the C2− and CN− maps closely match the secondary electron map shown in figure 3(B) while the P− signal was too weak to obtain any useful information from it. The O− and Si− maps show the presence of the glass substrate in the regions where the ion sputtering has removed the Au-Pd layer exposing the silica underneath. The most intense signals appear closest to the cell possibly due to freshly exposed glass surfaces where the cell previously had covered. These maps were also expected to show the location of the R-AuNPs due to the silica shell but this did not occur.
NIH-PA Author Manuscript
The Au− map shows the locations of a number of R-AuNPs in the sample. The nanoparticles do not appear as crisp as in the SEM image, due to decreased resolution and not all of them are visible as shown in figure 5. Comparing the SEM image with the NanoSIMS image, it is possible to locate and identify individual R-AuNPs in the NanoSIMS image but when multiple R-AuNPs are located together their signals appear to blur together making quantification difficult, if not impossible. Additionally, it is clear that not all of the R-AuNPs are visible in the NanoSIMS image, figure 5(A). This could likely occur because the RAuNPs visible in the SEM image, figure 5(B), could be deeper in the tissue and have not yet been exposed at the surface. Furthermore, the some of the R-AuNPs could have been sputtered away and are no longer present in the sample.
CONCLUSIONS NanoSIMS is a very useful technique for biological applications. However for locating, imaging and identifying inorganic nanoparticles in complex systems SEM is a superior technique. NanoSIMS is limited by a lower spatial resolution of about 50 nm whereas SEM can achieve resolutions down to 1 nm. The ability to differentiate between isotopes is a unique capability of the NanoSIMS but with inorganic nanoparticles like the R-AuNPs this is not an issue. The ability to identify the R-AuNPs using backscattered electron detection in the SEM allows for higher spatial resolution with increased depth penetration than NanoSIMS.
NIH-PA Author Manuscript
Acknowledgments Part of this research was conducted using the Cameca NanoSIMS 50L supported by the NSF under award #0922684. The work was funded by NCI Center for Cancer Nanotechnology Excellence GrantCCNE U54 U54CA151459 (S.S.G.).
REFERENCES 1. Zavaleta CL, Hartman KB, Miao Z, James ML, Kempen P, Thakor AS, Nielsen CH, Sinclair R, Cheng Z, Gambhir SS. Small. 2011; 7(15):2232–2240. [PubMed: 21608124] 2. Devaraj NK, Keliher EJ, Thurber GM, Nahrendorf M, Weissleder R. Bioconjugate chemistry. 2009; 20(2):397–401. [PubMed: 19138113] 3. Liu Z, Tabakman S, Sherlock S, Li X, Chen Z, Jiang K, Fan S, Dai H. Nano research. 2010; 3(3): 222–233. [PubMed: 21442006] Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
Kempen et al.
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NIH-PA Author Manuscript NIH-PA Author Manuscript
4. Roussel LY, Stokes DJ, Gestmann I, Darus M, Young RJ. presented at the Proc. SPIE. 2009 (unpublished). 5. Koh AL, Shachaf CM, Elchuri S, Nolan GP, Sinclair R. Ultramicroscopy. 2008; 109(1):111–121. [PubMed: 18995965] 6. Reimer L. Measurement Science and Technology. 2000; 11(12):1826. 7. Zhou, W.; Wang, ZL. Scanning microscopy for nanotechnology: techniques and applications. Springer; 2006. p. 1-40. 8. Wu B, Becker JS. International Journal of Mass Spectrometry. 2011; 307(1–3):112–122. 9. Pett-Ridge, J.; Weber, P. Microbial Systems Biology. Navid, A., editor. Vol. Vol. 881. Humana Press; 2012. p. 375-408. 10. Wells J, Kilburn MR, Shaw JA, Bartlett CA, Harvey AR, Dunlop SA, Fitzgerald M. Journal of Neuroscience Research. 2012; 90(3):606–618. [PubMed: 22038561] 11. Steinhauser ML, Bailey AP, Senyo SE, Guillermier C, Perlstein TS, Gould AP, Lee RT, Lechene CP. Nature. 2012 12. Kircher MF, de la Zerda A, Jokerst JV, Zavaleta CL, Kempen PJ, Mittra E, Pitter K, Huang R, Campos C, Habte F, Sinclair R, Brennan CW, Mellinghoff IK, Holland EC, Gambhir SS. Nature Medicine. 2012; 18(5):829–834. 13. Jokerst JV, Thangaraj M, Kempen PJ, Sinclair R, Gambhir SS. ACS Nano. 2012; 6(7):5920–5930. [PubMed: 22681633] 14. Keren S, Zavaleta C, Cheng Z, De La Zerda A, Gheysens O, Gambhir S. Proceedings of the National Academy of Sciences. 2008; 105(15):5844–5849. 15. Mulvaney SP, Musick MD, Keating CD, Natan MJ. Langmuir. 2003; 19(11):4784–4790. 16. Kempen PJ. Stanford University. 2012 17. Dykstra, MJ.; Reuss, LE. Biological electron microscopy: theory, techniques, and troubleshooting. Springer; 2003. p. 1-71.
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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1.
NIH-PA Author Manuscript
(A) Low magnification secondary electron image of a macrophage with a (B) higher magnification image shown R-AuNPs on the surface of the macrophage. (C) Low magnification backscattered electron image of a macrophage showing a number of bright RAuNPs all over the cell surface with a (D) higher magnification image showing the bright gold core of the R-AuNPs on the surface of a macrophage. Scale (A,C) = 2 µm (B,D) = 200 nm.
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Figure 2.
(A) Backscattered electron image taken in the SEM showing the cell analyzed in the NanoSIMS. (B) Higher magnification BSE image from the SEM of the boxed area in (A) showing the area directly analyzed for R-AuNPs in the NanoSIMS. R-AuNPs appear as bright areas in both (A) and (B). Scale (A) = 5 µm (B) = 1 µm.
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Figure 3.
(A) Secondary electron image acquired in the NanoSIMS of the same cell shown in figure 2(A). The decreased resolution is due to the use of the ion beam to generate the secondary electrons and the increased charging caused by the glass substrate. (B) Higher magnification secondary electron image of the area shown in figure 2(B) with no R-AuNPs visible. Scale (A) = 5 µm (B) = 500 nm.
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Figure 4.
Compositional maps for (A) C2− and (B) CN− showing the structure of the cell in this region, (C) O− and (D) Si− showing the glass regions exposed by the increased sputtering, (E) P− showing a weak signal where the cell is located and (F) Au− showing the presences of the R-AuNPs that have been exposed to the surface. Scale = 500 nm.
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Figure 5.
(A) NanoSIMS Au− map showing the location of the R-AuNPs indicating that not all the RAuNPs located in (B), the SEM image, are visible in the NanoSIMS image. Scale = 1 µm.
NIH-PA Author Manuscript Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
NIH-PA Author Manuscript
Published in final edited form as: Mater Res Soc Symp Proc. 2013 May 13; 1569: 157–163.
Advanced Characterization Techniques for Nanoparticles for Cancer Research: Applications of SEM and NanoSIMS for Locating Au Nanoparticles in Cells Paul J Kempen1, Chuck Hitzman2, Laura S Sasportas3, Sanjiv S Gambhir3, and Robert Sinclair1 1Department
of Material Science and Engineering, Stanford University, 496 Lomita Mall, Stanford, CA 94305-4034 U.S.A.
2Stanford
Nanocharacterization Laboratory, Stanford University, 476 Lomita Mall, Stanford, CA 94305-4035 U.S.A. 3Molecular
NIH-PA Author Manuscript
Imaging Program at Stanford, Department of Radiology, Stanford University, 318 Campus Drive, Stanford, CA 94305-5427 U.S.A.
Abstract The ability of nano secondary ion mass spectrometry (NanoSIMS) to locate and analyze Raman active gold core nanoparticles (R-AuNPs) in a biological system is compared with the standard analysis using the scanning electron microscope (SEM). The same cell with R-AuNPs on and inside the macrophage was analyzed with both techniques to directly compare them. SEM analysis showed a large number of nanoparticles within the cell. Subsequent NanoSIMS analysis showed fewer R-AuNPs with lower spatial resolution. SEM was determined to be superior to NanoSIMS for the analysis of inorganic nanoparticles in complex biological systems.
INTRODUCTION
NIH-PA Author Manuscript
With the continuing and growing use of nanoparticles for biological applications [1–3] it is becoming increasingly important to be able to accurately locate and characterize them on and within a cell. Owing to their small size it is necessary to utilize advanced characterization techniques to study them. The scanning electron microscope (SEM), capable of sub-nanometer spatial resolution [4] and good depth of field, is commonly utilized for this purpose [5]. Moreover, it is possible to distinguish materials by atomic number through the use of backscattered electron (BSE) imaging [6]. This atomic number sensitivity creates contrast in the image where higher atomic number inorganic nanoparticles appear bright against a dark low atomic number, organic matrix background. This enables the ready identification of inorganic nanoparticles both on the surface and in the cytoplasm of cell samples [7]. Recently there have been advances in secondary ion mass spectrometry (SIMS), combining SIMS with a focused scanning ion beam, allowing for the creation of high spatial resolution, ca 50 nm, compositional maps [8]. The NanoSIMS combines reasonably good spatial resolution with high atomic sensitivity, down to the ppm regime [8] and is powerful because
Kempen et al.
Page 2
NIH-PA Author Manuscript
it is capable of distinguishing between isotopes of the same material while analyzing up to seven distinct elements or species at the same time [9]. This technique has been increasingly utilized to study and analyze complex biological systems including the use of isotope labeling [10, 11]. Gold-core silica-shell nanoparticles (R-AuNPs) are promising as a multimodality surface enhanced Raman spectroscopy (SERS) and photoacoustic nanoparticle for diagnostic purposes [12–14]. R-AuNPs, produced by Oxonica Materials Inc (now owned by Cabot Corporation), consist of a 60 nm diameter gold core surrounded by a 30 nm thick silica shell. A monolayer of Raman active dye is adsorbed onto the surface of the gold core providing a surface enhancement effect that can improve the Raman signal intensity by many orders of magnitude [15]. SEM is an established technique for analyzing nanoparticles and has previously been utilized to locate and analyze R-AuNPs in a number of biological systems including T-cells [16] and brain tumors [12]. In this work SEM analysis of RAW264.7 human macrophages incubated with R-AuNPs was compared with NanoSIMS analysis of the same area to determine the viability of NanoSIMS as a technique to locate and characterize inorganic nanoparticles in biological systems.
NIH-PA Author Manuscript
EXPERIMENT R-AuNPs were incubated with RAW264.7 human macrophages grown on glass cover slips to induce cellular uptake of the nanoparticles. The cells were then fixed in a solution of 2.5% glutaraldehyde 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 1 hour at room temperature. The samples were then dehydrated in increasing concentrations of ethanol; 50%, 70%, 95% and 100% twice, at 20 minute intervals. The cells were then prepared for SEM and NanoSIMS analysis through critical point drying (CPD), creating a vacuum compatible sample while preserving the structure of the cells [17]. The glass cover slips were then placed on aluminum SEM stubs and coated with a thin layer of Au-Pd to improve conduction. The samples were then cross hatched with a diamond scribe to allow for co-localization between the SEM and the NanoSIMS.
NIH-PA Author Manuscript
The samples were imaged in the SEM using a FEI Magellan 400 XHR SEM operated at 15 kV with a probe current of 50 pA. After imaging in the SEM, the location was recorded by acquiring a low magnification montage image of the cover slip using the automated software in the Magellan and the sample was placed in the Cameca NanoSIMS 50L. Cesium+ primary ion beam bombardment and negative secondary ion detection were used to optimize the sensitivity for Au. The primary beam current at the sample was on the order of 1pA with an impact energy of 16 keV normal to the surface. Using the secondary electron signal generated by the scanning cesium, Cs+, ion beam the exact location was located. Composition maps for C2−, CN−, O−, Si−, P− and Au− were then generated to show the location of the R-AuNPs within the cell.
Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
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DISCUSSION NIH-PA Author Manuscript
Scanning Electron Microscopy
NIH-PA Author Manuscript
Scanning electron microscopy is a powerful technique for analyzing R-AuNPs on and inside cells. Secondary electron (SE) imaging provides excellent surface topography information, as shown in figure 1(a–b), while BSE imaging can provide elemental information about the sample as shown in figure 1(c–d). The surface structure is more identifiable in the SE images while the R-AuNPs are can clearly be identified on the surface of the macrophage, appearing as bright areas in the BSE image due to the high atomic number gold core. Figure 1 is representative of the types of images that can be obtained from the SEM. SEM analysis indicated that the R-AuNPs were located across the surface of the macrophages, making this a useful system for comparison with NanoSIMS. A BSE image of the cell that was analyzed using the NanoSIMS is shown in figure 2, with a higher magnification image of the area analyzed for R-AuNPs. This image shows a large number of nanoparticles in the area of interest on or in the cell. BSE imaging can image R-AuNPs that are not at the surface of the sample because the high energy electron beam can penetrate into the sample creating signal several hundred nanometers below the surface of the cell. Some of the R-AuNPs shown in figure 2(B) are located within the cell and not on the surface. This cell was chosen because of the large extension in the upper part region of the cell allowing for easier identification in the NanoSIMS. Nano Secondary Ion Mass Spectrometry
NIH-PA Author Manuscript
Locating the same cell in the NanoSIMS is a non-trivial endeavor due to differences in image formation and quality. There is an image inversion in the NanoSIMS that must be accounted for when navigating and locating a cell. Once this is taken into account, there are still large differences in image quality due to the ion beam. This beam has a much larger probe diameter, around 50 nm, than the electron beam in the SEM, approximately 1 nm, resulting in decreased surface resolution, shown in figure 3. Additionally, the ion beam quickly sputters the thin layer of Au-Pd on the surface of the sample, exposing the underlying cell and glass substrate leading to localized charging. The cell in figure 3(a), the same cell shown in figure 2, appears more blurred as a result of the decreased resolution. There are also bright regions in the image that do not correspond to R-AuNPs but are rather due to charging of the sample. This makes identification of the same region between the two instruments difficult. The unusual shape of this cell was the single identifiable feature between the two techniques. It took about four hours to locate this specific cell in the NanoSIMS. Once the cell was identified at low magnification, the region of the cell indicated in the white box in figure 3 was analyzed for the presence of C2−, CN−, O−, Si−, P− and Au−. Initially no gold was identified in this region. This happened because the R-AuNPs are coated with silica preventing the sputtering of the underlying gold and the nanoparticles were located within the cell in this region requiring the sputtering of the surface of the cell before the R-AuNPs would become visible. NanoSIMS is essentially a surface sensitive technique because ion generation and sputtering can only occur within the first 1–2 nm of the surface. After slowly sputtering the surface away for approximately 30 minutes to
Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
Kempen et al.
Page 4
expose the R-AuNPs, compositional maps for C2−, CN−, O−, Si−, P− and Au− were created, figure 4.
NIH-PA Author Manuscript
The composition maps show the location of the cell in the analyzed region as well as some of the R-AuNPs located in that region. The C2−, CN− and P− maps show the structure of the cell. Both the C2− and CN− maps closely match the secondary electron map shown in figure 3(B) while the P− signal was too weak to obtain any useful information from it. The O− and Si− maps show the presence of the glass substrate in the regions where the ion sputtering has removed the Au-Pd layer exposing the silica underneath. The most intense signals appear closest to the cell possibly due to freshly exposed glass surfaces where the cell previously had covered. These maps were also expected to show the location of the R-AuNPs due to the silica shell but this did not occur.
NIH-PA Author Manuscript
The Au− map shows the locations of a number of R-AuNPs in the sample. The nanoparticles do not appear as crisp as in the SEM image, due to decreased resolution and not all of them are visible as shown in figure 5. Comparing the SEM image with the NanoSIMS image, it is possible to locate and identify individual R-AuNPs in the NanoSIMS image but when multiple R-AuNPs are located together their signals appear to blur together making quantification difficult, if not impossible. Additionally, it is clear that not all of the R-AuNPs are visible in the NanoSIMS image, figure 5(A). This could likely occur because the RAuNPs visible in the SEM image, figure 5(B), could be deeper in the tissue and have not yet been exposed at the surface. Furthermore, the some of the R-AuNPs could have been sputtered away and are no longer present in the sample.
CONCLUSIONS NanoSIMS is a very useful technique for biological applications. However for locating, imaging and identifying inorganic nanoparticles in complex systems SEM is a superior technique. NanoSIMS is limited by a lower spatial resolution of about 50 nm whereas SEM can achieve resolutions down to 1 nm. The ability to differentiate between isotopes is a unique capability of the NanoSIMS but with inorganic nanoparticles like the R-AuNPs this is not an issue. The ability to identify the R-AuNPs using backscattered electron detection in the SEM allows for higher spatial resolution with increased depth penetration than NanoSIMS.
NIH-PA Author Manuscript
Acknowledgments Part of this research was conducted using the Cameca NanoSIMS 50L supported by the NSF under award #0922684. The work was funded by NCI Center for Cancer Nanotechnology Excellence GrantCCNE U54 U54CA151459 (S.S.G.).
REFERENCES 1. Zavaleta CL, Hartman KB, Miao Z, James ML, Kempen P, Thakor AS, Nielsen CH, Sinclair R, Cheng Z, Gambhir SS. Small. 2011; 7(15):2232–2240. [PubMed: 21608124] 2. Devaraj NK, Keliher EJ, Thurber GM, Nahrendorf M, Weissleder R. Bioconjugate chemistry. 2009; 20(2):397–401. [PubMed: 19138113] 3. Liu Z, Tabakman S, Sherlock S, Li X, Chen Z, Jiang K, Fan S, Dai H. Nano research. 2010; 3(3): 222–233. [PubMed: 21442006] Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
Kempen et al.
Page 5
NIH-PA Author Manuscript NIH-PA Author Manuscript
4. Roussel LY, Stokes DJ, Gestmann I, Darus M, Young RJ. presented at the Proc. SPIE. 2009 (unpublished). 5. Koh AL, Shachaf CM, Elchuri S, Nolan GP, Sinclair R. Ultramicroscopy. 2008; 109(1):111–121. [PubMed: 18995965] 6. Reimer L. Measurement Science and Technology. 2000; 11(12):1826. 7. Zhou, W.; Wang, ZL. Scanning microscopy for nanotechnology: techniques and applications. Springer; 2006. p. 1-40. 8. Wu B, Becker JS. International Journal of Mass Spectrometry. 2011; 307(1–3):112–122. 9. Pett-Ridge, J.; Weber, P. Microbial Systems Biology. Navid, A., editor. Vol. Vol. 881. Humana Press; 2012. p. 375-408. 10. Wells J, Kilburn MR, Shaw JA, Bartlett CA, Harvey AR, Dunlop SA, Fitzgerald M. Journal of Neuroscience Research. 2012; 90(3):606–618. [PubMed: 22038561] 11. Steinhauser ML, Bailey AP, Senyo SE, Guillermier C, Perlstein TS, Gould AP, Lee RT, Lechene CP. Nature. 2012 12. Kircher MF, de la Zerda A, Jokerst JV, Zavaleta CL, Kempen PJ, Mittra E, Pitter K, Huang R, Campos C, Habte F, Sinclair R, Brennan CW, Mellinghoff IK, Holland EC, Gambhir SS. Nature Medicine. 2012; 18(5):829–834. 13. Jokerst JV, Thangaraj M, Kempen PJ, Sinclair R, Gambhir SS. ACS Nano. 2012; 6(7):5920–5930. [PubMed: 22681633] 14. Keren S, Zavaleta C, Cheng Z, De La Zerda A, Gheysens O, Gambhir S. Proceedings of the National Academy of Sciences. 2008; 105(15):5844–5849. 15. Mulvaney SP, Musick MD, Keating CD, Natan MJ. Langmuir. 2003; 19(11):4784–4790. 16. Kempen PJ. Stanford University. 2012 17. Dykstra, MJ.; Reuss, LE. Biological electron microscopy: theory, techniques, and troubleshooting. Springer; 2003. p. 1-71.
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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1.
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(A) Low magnification secondary electron image of a macrophage with a (B) higher magnification image shown R-AuNPs on the surface of the macrophage. (C) Low magnification backscattered electron image of a macrophage showing a number of bright RAuNPs all over the cell surface with a (D) higher magnification image showing the bright gold core of the R-AuNPs on the surface of a macrophage. Scale (A,C) = 2 µm (B,D) = 200 nm.
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Figure 2.
(A) Backscattered electron image taken in the SEM showing the cell analyzed in the NanoSIMS. (B) Higher magnification BSE image from the SEM of the boxed area in (A) showing the area directly analyzed for R-AuNPs in the NanoSIMS. R-AuNPs appear as bright areas in both (A) and (B). Scale (A) = 5 µm (B) = 1 µm.
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Figure 3.
(A) Secondary electron image acquired in the NanoSIMS of the same cell shown in figure 2(A). The decreased resolution is due to the use of the ion beam to generate the secondary electrons and the increased charging caused by the glass substrate. (B) Higher magnification secondary electron image of the area shown in figure 2(B) with no R-AuNPs visible. Scale (A) = 5 µm (B) = 500 nm.
NIH-PA Author Manuscript Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
Kempen et al.
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NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 4.
Compositional maps for (A) C2− and (B) CN− showing the structure of the cell in this region, (C) O− and (D) Si− showing the glass regions exposed by the increased sputtering, (E) P− showing a weak signal where the cell is located and (F) Au− showing the presences of the R-AuNPs that have been exposed to the surface. Scale = 500 nm.
NIH-PA Author Manuscript Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
Kempen et al.
Page 10
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 5.
(A) NanoSIMS Au− map showing the location of the R-AuNPs indicating that not all the RAuNPs located in (B), the SEM image, are visible in the NanoSIMS image. Scale = 1 µm.
NIH-PA Author Manuscript Mater Res Soc Symp Proc. Author manuscript; available in PMC 2014 October 31.
