Interrogating Cells, Tissues, and Live Animals with New Generations

Feb 8, 2017 - Interrogating Cells, Tissues, and Live Animals with New Generations of Surface-Enhanced Raman Scattering Probes and Labels. Janina Kneip...
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Interrogating Cells, Tissues, and Live Animals with New Generations of Surface-Enhanced Raman Scattering Probes and Labels Janina Kneipp* Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Strasse 2, 12489 Berlin, Germany ABSTRACT: Surface-enhanced Raman scattering (SERS) probes and labels exploit surface-enhanced Raman signatures of specifically chosen reporter molecules and intrinsic biomolecules in the plasmonic near field of gold and silver nanostructures. They offer several advantages over other optical labels and sensors with respect to sensitivity and selectivity, mobility, and biocompatibility. Here, the multifunctionality and versatility of SERS labels that come from the vibrational spectroscopic information and their ability to act as nanoprobes of a biological environment are discussed. Surface-enhanced Raman scattering probes have the potential to become the next-generation sensor technology for monitoring cells and tissues. They improve our understanding of cellular function and will play a major role in future theranostic applications. fields, which occur due to resonances between excitation and scattered field and surface plasmons, provide the main mechanism for the SERS effect. In addition, a second, socalled chemical enhancement mechanism, based on interaction of the molecule with the metal nanostructures, can increase the Raman cross-section of the adsorbed molecule. Plasmonic field enhancement can result in more than 10 orders of magnitude signal enhancement, whereas chemical enhancement can contribute an enhancement factor of 10−103. If the excitation light is in resonance with an electronic transition in the molecule, additional enhancement is observed due to resonant Raman scattering (SERRS) (Figure 1). The strong SERRS signal can serve as a spectroscopic signature of a label in a similar way as fluorescence light does. A SERS (or SERRS) label, sometimes also referred to as a SERS tag, typically consists of gold or silver nanoparticles and a reporter molecule that provides a characteristic Raman spectrum (Figure 2A). The whole label can be encapsulated in glass or polymer.2 The advantages of these coated SERS labels include physical robustness, stable signals, and immunity to their biological and chemical environments. To direct the SERS label to its biomolecular target, it can be functionalized using a targeting unit, such as an antibody, an oligonucleotide, a peptide, or a substrate binding to a specific receptor. As an advantage compared to fluorescence labels, great multiplexing capabilities of SERRS labels, resulting from the many narrow lines in the vibrational spectra, have been emphasized ever

SURFACE-ENHANCED RAMAN SCATTERING LABELS AND THEIR CAPABILITIES Surface-enhanced Raman scattering, the strong enhancement of the inelastic scattering (Raman) process (Figure 1), is observed when molecules reside in close proximity to plasmonic (typically gold or silver) nanostructures.1 High local optical

Figure 1. Spontaneous Raman scattering processes that are used in surface-enhanced Raman scattering (SERS) labels and probes. (A) Nonresonant Raman scattering (Stokes and anti-Stokes), (B) resonant Raman scattering, for surface-enhanced resonance Raman scattering (SERRS) labels, and (C) hyper-Raman scattering, for two-photon excited surface-enhanced hyper-Raman scattering (SEHRS). © 2017 American Chemical Society

Published: February 8, 2017 1136

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analogy to two different fluorescence spectra of two fluorophores, for the detection of micrometastases in ovarian cancer in whole animals.14 The two molecules IR780 and IR140, excited with a near-infrared laser, act as reporter molecules in the two respective SERRS labels. As plasmonic moieties, they contain gold nanostars that can provide high electromagnetic field enhancement for SERS.15,16 The authors combine single tumor cell detection by ratiometric analysis of SERRS label signatures with application of the labels in live animals and ex vivo imaging.14 Since the microscopic metastases that are targeted within the peritoneal surface are not connected to the circulation, the SERRS labels are injected directly into the peritoneum of the live animals. They reach their target, as they carry antifolate-receptor (αFR) antibodies specific for the folate receptor that is overexpressed in the ovarian cancer cells. As reported in the paper, the antibodies, rather than folate itself, were chosen to target the folate receptor with the labels, due to low and unspecific labeling with folate-directed tags.14 Recent work by Fasolato et al., who used noncapsulated SERS labels consisting of gold nanoparticles coated with 4-aminothiphenol and folate to distinguish between tumor cells expressing folate receptors (FRs), tumor cells without FRs, and normal cells in vitro, provides quantitative SERS evidence of low, nonspecific binding of folate-directed SERS labels to other folate transporters than FRs.17 This work illustrates that to detect the minute amounts of tumor cells in vivo in the microscopic metastases, the high sensitivity of SERRS must be accompanied by high selectivity of the targeting unit, and the specific interactions of the latter need to be verified upon attaching it to a SERRS label. Considering the sensitivity of SERRS labels, the Raman signal from the reporter molecules can be at least that of the fluorescence signal obtained with the same number of reporter molecules.18 The plasmonic nanostructure plays a key role for the performance of the SERS label, as it determines its signal strength. Further engineering of nanoparticle-reporter hybrids can make them brighter and make approaches such as the one reported by Oseledchyk et al.14 even more sensitive. A basic advantage of SERS labels is that a molecule can go through more Raman cycles than fluorescence cycles per time interval, due to shorter vibrational relaxation times compared to electronic relaxation times. Therefore, the number of Raman photons per unit time that can be emitted by a reporter molecule can be higher than the number of fluorescent photons. Moreover, SERRS studies have shown that the attachment of a molecule to a metal nanostructure improves its photostability19 and, therefore, the total outcome of SERRS photons of a dye molecule can be higher than the total number of fluorescence photons. Specific advantages for bioprobing come from the fact that SERS also works well as a nonresonant process (Figure 1). If no resonant excitation is required, many labels can be used at the same excitation wavelength. There, excitation can be chosen in the near-IR wavelength range, where relatively low-energy photons prevent photodecomposition of the samples, the offresonance excited reporter molecules are stable, and autofluorescence of tissues is weak. Despite these essential methodological advantages, a SERS label carries out the same functions as a fluorescence tagthat is, highlighting and imaging biological structures based on the optical signature of a reporter molecule. Merely adding their improved multiplexing and photostability to the slightly higher brightness may leave us with doubts about SERRS labels

Figure 2. Molecular imaging in tissues using surface-enhanced Raman scattering (SERS) labels and probes. (A) Schematic of a SERS label and different types of SERS probes that have been reported. Different reporter functions are indicated with starshaped symbols of different colors. Some SERS probes do not carry a reporter. Targeting can be achieved using different kinds of targeting units, such as antibodies, nuclear localization sequences, oligonucleotides, or substrates of specific receptors. (B) Interaction in a tissue occurs at the microscopic, subcellular level, and the compartments of the cell, its surface, and the interstitium can be probed. In principle, because of the multiplexing capabilities, several different probes with different targeting can be applied simultaneously. Abbreviations: Nu, nucleus, En, endosome.

since, as they yield good separation of spectral fingerprints, e.g., by multivariate statistical tools.3 At their advent more than 15 years ago,4 the first SERRS labels were proposed for multiplexed detection of proteins and nucleotides in microarrays5,6 or in immunoassays.7 Meanwhile, SER(R)S labeling of post-mortem excised tumor tissues,8 of isolated living tumor cells,9 and of tumors in vivo10 became feasible, along with proof-of-principle demonstrations that different SERRS labels can be detected in live animals.11 The high physicochemical stability and the stability of the spectral signatures of a particular encapsulated SERRS label enables its quantification,12,13 and, hence, quantification of its biomolecular target.

In this issue of ACS Nano, Oseledchyk et al. report using labels that employ the SERRS spectra of two molecules, in analogy to two different fluorescence spectra of two fluorophores, for the detection of micrometastases in ovarian cancer in whole animals. In this issue of ACS Nano, Oseledchyk et al. report using labels that employ the SERRS spectra of two molecules, in 1137

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outperforming fluorescence tags. However, Raman spectroscopic information on the molecules residing in the plasmonic near-field, that is, reporter molecules and/or intrinsic biomolecules, provides convincing evidence that the capabilities of SERS labels extend far beyond those of bright optical markers.

ROS that is responsible for killing pathogens in leukocytes, and glutathione (GSH), an important antioxidant related to oxidative stress in cells and tissues were monitored in live cells in real-time with SERS probes carrying 4-mercaptophenol.26 In Raman spectra, the ratio of the anti-Stokes-to-Stokes signals, originating from the molecules in the vibrational excited and ground states, respectively, is determined by the Boltzmann distribution. Therefore, it can be related to the temperature of the molecules. In SERS, processes other than temperature can also affect the anti-Stokes and Stokes signal intensities and can give rise to deviations in the ratio between them. Provided that the processes that disturb temperature-dependent anti-Stokesto-Stokes ratios are understood for the reporter molecule and the plasmonic nanostructure of a particular SERS probe, temperature sensing with SERS probes is possible.27,28

Raman spectroscopic information on the molecules residing in the plasmonic near-field, that is, reporter molecules and/or intrinsic biomolecules, provides convincing evidence that the capabilities of SERS labels extend far beyond those of bright optical markers.

Directly probing the structure and composition of the intrinsic molecules in cells and tissues using their own spectra is one of the most exciting aspects of SERS.

PROBING THE CHEMICAL ENVIRONMENT BEYOND SERS TAGGING The spectroscopic signatures of reporter molecules, as well as SERS spectra from their biological environment, transform SERS labels into SERS probes of cellular and tissue chemistry. The vibrational spectrum of the reporter molecule provides structural information about the reporter itself and its interaction with the plasmonic nanostructures, the shell, the targeting units, or molecules in the biological environment. Depending on reporter molecules interacting with the bioorganic molecules in the cellular or tissue surroundings, a SERS label acts as a sensitive nanoprobe. Compared to other optical sensors, such SERS nanosensors yield information using the relative signals of spectrally narrow pairs of Raman lines in the same reporter spectrum. This information enables quantitative measurements without any correction regarding cellular background absorption and emission signals. As a popular example, reporter molecules that have pHsensitive Raman spectra enable pH imaging in single live cells at subendosomal resolution and monitoring of pH distributions in cell populations.20 The sensitivity of the pH nanoprobes relies on the structural change of the reporter molecules upon protonation/deprotonation in the cellular surroundings.21 The interactions of the reporter with the plasmonic nanostructure are important as well. Approaches that can monitor these interactions, such as surface-enhanced hyper-Raman scattering (SEHRS) probes (Figure 1) improve the performance of the pH sensors and extend their operational range far into acidic values.22 The ability to image the acidic pH of endosomal/ phagosomal and lysosomal structures in cells has great significance for elucidating interactions of pathogens and for therapeutic approaches,23,24 e.g., pH-responsive drug targeting. In tissues, pH regulation in the interstitium plays a major role, not only in many critical illnesses or in tumor microenvironments but also during normal physiological operation, e.g., exercise-induced acidosis, brain function, embryogenesis, and many more. Monitoring interstitial and intracellular pH in vivo, along with other molecular parameters by SERS probes is one of the main goals of current research in SERS bioprobes (Figure 2B, red nanoprobes). Similarly, H2O2, a major reactive oxygen species (ROS) and second messenger can be quantified using a reporter consisting of 4-mercaptophenylboronic ester attached to gold nanorods.25 Other molecular species, in particular hypochlorite (ClO−), an

Directly probing the structure and composition of the intrinsic molecules in cells and tissues using their own spectra is one of the most exciting aspects of SERS. It requires interactions of the bioenvironment with the plasmonic parts of the nanoprobes. This strategy is applied with SERS probes that contain reporter molecules that render the surfaces of the plasmonic nanostructures accessible to cellular molecules29 or that do not carry reporter molecules at all30 (see Figure 2 for schematics of both types of SERS probes). Images of the samples, often cultured cells, show the distributions of the SERS probes, and their spectra can indicate which molecules interact with the plasmonic nanostructures in a particular experiment and how they interact. For example, cargo that is transported by gold nanoparticles in order to be delivered into cells as a therapeutic can be tracked, based on its spectrum, until it is “dumped” from the plasmonic nanostructure.31,32 Likewise, the biomolecular corona of nanostructures inside the living cell can be characterized over time,33 and the interaction of cultured cells with nanomaterials that have different surface properties can be investigated. As an example, core−shell structures with a plasmonic core that mimic the surface of silica nanoparticles, termed BrightSilica, have different molecules interacting with their surface depending on the cell type and the associated route of uptake.34 The cell cycle has been characterized by SERS probes targeted to the nuclei,32 and cellular transport pathways can be followed by following intracellular vesicles that carry SERS probes35 (see schematic in Figure 2B). Furthermore, SERS nanoprobes can be used to image metabolically important species in living cells, such as adenosine monophosphate in macrophages,30 and even in whole microscopic animals, e.g., lipid storage granules in the intestine of the microscopic nematode Caenorhabditis elegans (C. elegans),36 or trehalose, a disaccharide that enables cryptobiosis in tardigrades.37 In addition to the SERS spectral contributions that come from intrinsic biomolecules, the spectral signature of reporter molecules incorporated in a SERS probe (Figure 2, blue stars) can be useful if several types of probes are used at the same 1138

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of plasmonic nanostructures in cells,34 have been proposed to optimize probe targeting. Furthermore, high-resolution X-ray nanotomography can give precise information about the localization of SERS nanoprobes with respect to cellular organelles (Figure 3).33

time, especially when they interact and are contained in different cellular compartments.3 In addition to seeing the molecules in their environment, the probe can be identified by the characteristic SERS spectrum of the reporter, and both kinds of information can be retrieved in multivariate data analysis.

OUTLOOK AND CHALLENGES: FROM CULTURED CELLS TO LIVE ANIMALS Surface-enhanced Raman scattering probes and labels offer several advantages over other optical probes with respect to versatility, sensitivity and selectivity, and biocompatibility. Most of the exciting current practical applications of SERS nanoprobing seem to occur for advanced sensing and imaging in the biomedical field. Future challenges include the development of multifunctional SERS probes with optimized plasmonic nanostructures as basic building blocks and their targeting in whole organisms. Plasmonic structures as key components of SERS probes can offer high local optical fields for both sensitive diagnostic probing with SERS and efficient therapeutic tools, e.g., SERS in combination with plasmon-supported, improved light-based therapies.38 Particularly for applications in biological objects, probes based on two-photon excitation benefit from excitation at longer wavelengths in the near-IR. Several two-photon optical probes are used in biomedical imaging, among them second harmonic generation and two-photon fluorescence. The concept of SERS probes and labels can also be extended to two-photon excitation using SEHRS. With effective cross sections that can be higher than typical two-photon cross sections of fluorophores,39 SEHRS labels and probes have generated spectra from cultured cells39 and are well-suited for applications in tissues. Because of selection rules that differ from those of SERS, they deliver additional chemical information and display improved selectivity and specificity. Inside living cells or in tissue, the spectra measured with SERS probes give information on biological structures and processes from molecular perspectives, with extremely high lateral resolution from nanoscaled volumes. Because the nanoscopic SERS probes and labels are used to interrogate microscopic (cells and tissues) or macroscopic structures (whole organs or animals), depending on their dose, distribution, and detection modality, they can function as both individual nanosensors or ensembles that provide “average signals”. Especially in macroscopic imaging, where heterogeneity at the microscopic level due to histological or cellular substructures is not evident, the performance of a SERS label will be determined by the selectivity of its targeting unit. Understanding the interactions of SERS probes and labels with their environment at the cellular level will remain indispensable. This need to understand interactions is also true for microscopic sensing and imaging: interpreting SERS images or spectra that come from SERS probes inside individual cells requires understanding of their interaction at the level of the cellular ultrastructure. Microscopic examination of tissues parallel to in vivo probing is critical for validation but even more for understanding the interactions of the probes at the tissue and cellular level and for optimizing targeting. Combinations of Raman imaging with other microscopies, e.g., dark field microscopy32 or spatially resolved laser-ablation inductively coupled mass spectrometry imaging, the latter of which is capable of absolute quantification

Figure 3. Localization of typical surface-enhanced Raman scattering (SERS) labels consisting of silver nanoparticles, para-aminothiophenol as a reporter molecule, and a silica shell by nanoscale X-ray tomography. Parts A−D show different slices of a tomographic reconstruction of a vitrified J774 macrophage cell after incubation with the SERS labels in cell culture medium for 24 h. Uptake occurred by endocytosis, the absence of a targeting unit results in accumulation in endosomes/lysosomes The particles are enclosed in vesicular structures in the proximity of mitochondria or the cell nucleus. All images were acquired with a 25 nm zone plate (9.8 nm pixel size). A pixel binning of 2 × 2 was used. Scale bars: 1 μm. Abbreviations: N, nucleus; NM, nuclear membrane; M, mitochondrion; V, vesicle; PM, plasma membrane. Adapted with permission from ref 34. Copyright 2014 Wiley.

For basic investigations of SERS probes and labels in vivo, small nonvertebrate model organisms, such as C. elegans are interesting, dynamic samples, providing different tissue types as well as uptake and processing pathways.36 Penetration depths of the excitation and scattering light can transit the whole animal. The Raman microscopic experiments enable retrieval of both, histological information and data on subcellular localization in the same experiment and provide insight into the possible routing of nanoprobes and labels. For studies of larger animals, for example, small rodents in the laboratory, Raman imaging of the whole organism requires several different types of experiments. While confocal approaches can be applied for subcutaneous regions10 or in ex vivo measurements as discussed by Oseledchyk et al.,14 spatially offset Raman scattering (SORS) offers a promising approach for probing deeper layers of tissue in the body40 and has been combined with SERS in a number of experiments (SESORS), ranging from the recovery of SERRS signals from samples on the order of 45−50 mm thickness41 over transcutaneous in vivo glucose detection by implanted sensors in living rats42 to the quantification of diluted SERS labels through bone.43 As demonstrated recently for porcine skin 1139

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samples, SESORS, in principle, enables temperature measurements by subsurface SERS probes in turbid media.28 Utilizing the progress that has been made in the design of SERRS labels and probes, the application of SERS probes is about to revolutionize bioanalysis and future theranostic applications. The first proofs-of-principle are promising and will pave the way for subsurface, noninvasive SERS monitoring of cell and tissue parameters with exciting opportunities for in vivo molecular imaging.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Janina Kneipp: 0000-0001-8542-6331 Notes

The author declares no competing financial interest.

ACKNOWLEDGMENTS The author acknowledges funding by ERC Grant No. 259432 MULITBIOPHOT and by DFG FOR 2177/1 InCheM (Grant Kn557/12-1). REFERENCES (1) Kneipp, J.; Kneipp, H.; Kneipp, K. SERS - A Single-Molecule and Nanoscale Tool for Bioanalytics. Chem. Soc. Rev. 2008, 37, 1052− 1060. (2) Doering, W. E.; Piotti, M. E.; Natan, M. J.; Freeman, R. G. SERS as a Foundation for Nanoscale, Optically Detected Biological Labels. Adv. Mater. 2007, 19, 3100−3108. (3) Matschulat, A.; Drescher, D.; Kneipp, J. Surface-Enhanced Raman Scattering Hybrid Nanoprobe Multiplexing and Imaging in Biological Systems. ACS Nano 2010, 4, 3259−3269. (4) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903−4908. (5) Cao, Y. C.; Jin, R. C.; Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Raman Dye-Labeled Nanoparticle Probes for Proteins. J. Am. Chem. Soc. 2003, 125, 14676−14677. (6) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection. Science 2002, 297, 1536−1540. (7) Grubisha, D. S.; Lipert, R. J.; Park, H. Y.; Driskell, J.; Porter, M. D. Femtomolar Detection of Prostate-Specific Antigen: An Immunoassay Based on Surface-Enhanced Raman Scattering and Immunogold Labels. Anal. Chem. 2003, 75, 5936−5943. (8) Schlücker, S.; Küstner, B.; Punge, A.; Bonfig, R.; Marx, A.; Ströbel, P. Immuno-Raman Microspectroscopy: In Situ Detection of Antigens in Tissue Specimens by Surface-Enhanced Raman Scattering. J. Raman Spectrosc. 2006, 37, 719−721. (9) Pallaoro, A.; Braun, G. B.; Moskovits, M. Quantitative Ratiometric Discrimination Between Noncancerous and Cancerous Prostate Cells Based on Neuropilin-1 Overexpression. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16559−16564. (10) Qian, X. M.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. M. In Vivo Tumor Targeting and Spectroscopic Detection with SurfaceEnhanced Raman Nanoparticle Tags. Nat. Biotechnol. 2008, 26, 83− 90. (11) Keren, S.; Zavaleta, C.; Cheng, Z.; de la Zerda, A.; Gheysens, O.; Gambhir, S. S. Noninvasive Molecular Imaging of Small Living Subjects Using Raman Spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5844−5849. (12) Shaw, C. P.; Fan, M. K.; Lane, C.; Barry, G.; Jirasek, A. I.; Brolo, A. G. Statistical Correlation Between SERS Intensity and Nanoparticle Cluster Size. J. Phys. Chem. C 2013, 117, 16596−16605. 1140

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(33) Drescher, D.; Guttmann, P.; Buchner, T.; Werner, S.; Laube, G.; Hornemann, A.; Tarek, B.; Schneider, G.; Kneipp, J. Specific Biomolecule Corona is Associated with Ring-Shaped Organization of Silver Nanoparticles in Cells. Nanoscale 2013, 5, 9193−9198. (34) Drescher, D.; Zeise, I.; Traub, H.; Guttmann, P.; Seifert, S.; Buchner, T.; Jakubowski, N.; Schneider, G.; Kneipp, J. In Situ Characterization of SiO2 Nanoparticle Biointeractions Using BrightSilica. Adv. Funct. Mater. 2014, 24, 3765−3775. (35) Ando, J.; Fujita, K.; Smith, N. I.; Kawata, S. Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles. Nano Lett. 2011, 11, 5344−5348. (36) Charan, S.; Chien, F.-C.; Singh, N.; Kuo, C.-W.; Chen, P. Development of Lipid Targeting Raman Probes for in Vivo Imaging of Caenorhabditis elegans. Chem. - Eur. J. 2011, 17, 5165−5170. (37) Kneipp, H.; Mobjerg, N.; Jorgensen, A.; Bohr, H. G.; HelixNielsen, C.; Kneipp, J.; Kneipp, K. Surface Enhanced Raman Scattering on Tardigrada towards Monitoring and Imaging Molecular Structures in Live Cryptobiotic Organisms. J. Biophoton. 2013, 6, 759− 764. (38) Ali, M. R. K.; Panikkanvalappil, S. R.; El-Sayed, M. A. Enhancing the Efficiency of Gold Nanoparticles Treatment of Cancer by Increasing their Rate of Endocytosis and Cell Accumulation Using Rifampicin. J. Am. Chem. Soc. 2014, 136, 4464−4467. (39) Kneipp, J.; Kneipp, H.; Kneipp, K. Two-Photon Vibrational Spectroscopy for Biosciences Based on Surface-Enhanced HyperRaman Scattering. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 17149− 17153. (40) Matousek, P. Deep Non-Invasive Raman Spectroscopy of Living Tissue and Powders. Chem. Soc. Rev. 2007, 36, 1292−1304. (41) Stone, N.; Kerssens, M.; Lloyd, G. R.; Faulds, K.; Graham, D.; Matousek, P. Surface Enhanced Spatially Offset Raman Spectroscopic (SESORS) Imaging - The Next Dimension. Chem. Sci. 2011, 2, 776− 780. (42) Ma, K.; Yuen, J. M.; Shah, N. C.; Walsh, J. T.; Glucksberg, M. R.; Van Duyne, R. P. In Vivo, Transcutaneous Glucose Sensing Using Surface-Enhanced Spatially Offset Raman Spectroscopy: Multiple Rats, Improved Hypoglycemic Accuracy, Low Incident Power, and Continuous Monitoring for Greater than 17 Days. Anal. Chem. 2011, 83, 9146−9152. (43) Sharma, B.; Ma, K.; Glucksberg, M. R.; Van Duyne, R. P. Seeing through Bone with Surface-Enhanced Spatially Offset Raman Spectroscopy. J. Am. Chem. Soc. 2013, 135, 17290−17293.

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