Advances in Biomedical Raman Microscopy - Analytical Chemistry

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Advances in Biomedical Raman Microscopy Karen A. Antonio, and Zachary D. Schultz Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Nov 2013 Downloaded from http://pubs.acs.org on November 13, 2013

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Analytical Chemistry

Advances in Biomedical Raman Microscopy Karen A. Antonio and Zachary D. Schultz* University of Notre Dame Department of Chemistry and Biochemistry Notre Dame, IN 46556 *Corresponding author email: [email protected] The vibrational modes of molecules provide an intrinsic contrast mechanism for identifying components in cells, tissues and organisms. When excited by a laser, Raman scattered photons can be spatially correlated providing images of biomolecule distributions. The universality (all particles scatter) of Raman scattering enables in vitro and in vivo characterization. A variety of techniques have emerged based on conventional, surface enhanced, and nonlinear Raman methods that are successfully addressing biomedical challenges. Here we review the advances in Raman microscopy relevant to biomedical applications, focusing on research reported in the literature between 20112013. The ability to visualize a sample and understand the molecular composition and interactions that are occurring has made optical microscopy an invaluable tool for biomedical research. High-resolution optical microscopy has been dominated by fluorescence imaging due to the large signals generated by a vast collection of fluorophores over the years. However, Raman microscopy has been used for biomedical microscopy since 1979,1 when Raman spectra were acquired from a tissue sample in the view of an optical microscope. The different vibrational modes associated with different biomolecules provide intrinsic contrast that can elucidate both composition and interactions that occur. The peaks that arise from different functional groups can identify molecules, but the unique spectrum that arises from the combination of molecules in an organelle, cell or tissue can provide a characteristic pattern that enables classification. In the past 30 years, Raman has emerged as a sensitive probe of chemical composition. Advances in instrumentation, methodology, and data analysis have enabled Raman microscopy in a variety of applications from cellular analysis in vitro to in vivo clinical imaging. From advances in spontaneous Raman, to nanoparticle enhancements, and nonlinear Raman techniques, the use of Raman spectroscopy shows excellent prospects for a wide range of laboratory and clinical uses. 1. Spontaneous Raman Microscopy The inelastic scattering of a monochromatic light source, spontaneous Raman scattering, continues to be employed for biomedical applications, from monitoring the distribution of biomolecules in cells to providing chemical contrast for histopathology of cancer biopsies. When molecules have sufficiently large Raman cross-sections, or are present in significant concentration, these molecules can be mapped to determine how localization changes within cells and tissue. The principal advantage to using Raman for

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these applications is that chemical contrast is obtained in cells and tissues without the use of chemical labels,2 making Raman a powerful biomedical diagnostic.3 Recent examples include monitoring the distribution of intrinsic molecules in cells and tissues, studying drug delivery, and analyzing cancer biopsies and other tissue. The development of new instrumentation, methods, and data analysis has also provided new capabilities that extend the utility of Raman microscopy. Monitoring biomolecule concentrations within cells continues to be an important application of Raman microscopy. The localization and release of cytochrome c during apoptosis was reported by Fujita and coworkers by monitoring 750 cm-1 Raman band of cytochrome c.4 The mapped distribution of cytochrome c was distinctly observed in relation to the lipids and proteins in the cell as evident in Raman maps at 2857 cm-1 and

Figure 1. The 3D reconstruction of a EA.hy 926 cell from Raman images by (A) K-means cluster analysis using the Manhattan distance and 7 chemical clusters (red-necleolus; blue-small organelles, such as mitochondria, endoplasmic reticulum; brown-nucleoli; beige-cytoplasm; green-cell membrane; turquoisemostly cell membranes with adhesion proteins; orange-mostly adhesion proteins). (B) The average Raman spectra from 600-1800 cm-1 associated with chemical clusters are shown. (C) shows the cross-section of 3D image. (D) Top view of reconstructed 3D image is depicted. Reprinted with permission from K. Majzner, A. Kaczor, N. Kachamakova-Trojanowska, A. Fedorowicz, S. Chlopicki, M. Baranska, Analyst 2013, 138. 603-610. © 2013 Royal Society of Chemistry

1684 cm-1, respectively. A significant change in the distribution of cytochrome c was observed correlating with apoptosis. Other examples of monitoring the distribution of biomolecules in cells include identifying chemical signals that enable one to monitor the


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differentiation status in developing Ciona intestinalis embryos,5 and direct observation of cholesterol and unsaturated lipid distributions in the outer segment of rod photoreceptors.6 The ability to discriminate biochemical composition by Raman microscopy has resulted in recent reports of 3D Raman imaging. Organelle organization based on biochemical composition was reported in cells as shown in Figure 1.7 While the spectrum of pure components can be difficult to discern, the combinations of Raman bands provide a spectral signature associated with, for example, organelle type. In cytology samples, differences in nuclear shape, volume, and cell-cell distance were determined without altering the samples, thus preserving the integrity of the samples for further study.8 The uptake and distribution of drugs and drug-delivery vehicles has also been studied by conventional Raman spectroscopy. Studies on skin have examined the penetration of deuterated water and carotene into the stratum corneum.9 Differences in the Raman spectra of human and porcine hair follicles suggested use for drug-delivery studies.10 Resonance Raman detection of carotene was able to determine subcellular release from nanoparticle delivery systems.11 Similarly, drug delivery from cationic liposomes was also monitored.12 The release of doxorubicin form polymeric nanoparticles showed changes in the DNA bands within the nuclei of cells over time.13 Additionally, the antiobiotic Clofazimine was shown to accumulate in the mitochondria; interestingly, the Raman spectra provided additional information that the antibiotic was in an amorphous, rather than crystalline, state.14 There continues to be research into using Raman microscopy for histopathology. It was reported that Raman has greater success than conventional staining for diagnosing skin cancer melanoma and carcinoma,15 as well as the ability to distinguish between benign and malignant cancer lesions.16 Changes in the size of the nucleus during the cell cycle and proliferation of breast cancer cells suggested a diagnostic marker for cancer.17 Primary brain tumors studied by Raman imaging found correlations between malignancy and cell density,18 and similarly, Raman spectra could distinguish between normal and cancer mucosal tissue.19 In addition to cancer, other diseases have been studied with Raman microscopy. It was shown that Raman could identify cysteine crystals that accumulate in the body associated with the metabolic disorder nephropathic cystinosis.20 Studies revealed that aortic valve calcification showed calcium enrichment but not spectroscopic signatures characteristic of mineralization.21 Raman microscopy was able to discriminate between cholesterol esters, cholesterol and triglycerides in atherosclerotic plaques in rabbits.22 Studies of dental materials showed chemical differences in ceramic properties,23 and the role of hydration was studied in the degradation of other implants and scaffold materials.24 The progress noted above was accomplished through development of better and more efficient instrumentation, such as those noted in a recent review.25 Recent advances in instrumentation suggest continued growth in the use of Raman microscopy. The use fiber-optic coupled Raman microspectroscopy enabled monitoring of the extracellular matrix in a three-dimensional tissue scaffold for porous cartlidge growth.26 Light sheet Raman microscopy was used to monitor nucleic acids that were prominent in spectra obtained from a living fish, suggesting possibilities for tracking differentiation during development.27 Approaches, such as spatially offset Raman spectroscopy (SORS) continue


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to show utility for depth profiling in biomedical studies,28 including monitoring collagen to mineral ratios relevant to bone fracture,29 or tumor margins in tissue.30 The combination of Raman microscopy with other optical imaging techniques has been explored, such the combination with reflectance imaging to classify cancer cells 31 and

Figure 2. (A) The structure of thymidine species is shown noting the location of the alkyne marker functional group. (B) shows the Raman spectra of thymidine species with a representative cellular spectrum. The alkyne stretch frequency is clearly observed at 2122 cm-1. (C) Time-point composite Raman images of HeLa cells without 5-ethynyl-2’-deoxyuridine (EdU) serve as a control for comparison with cells treated with EdU for 3, 9, 12, 14, and 21 hours. The images show the distribution of cytochrome c at 729 cm-1 (blue), EdU at 2123 cm-1 in the nucleus (red), and protein at 2849 cm-1 (green). Adapted and reproduced from H. Yamakoshi, K. Dodo, M. Okada, J. Ando, A. Palonpon, K. Fujita, S. Kawata, M. Sodeoka, J. Am. Chem. Soc. 2011, 133. 6102-6105. © 2012 American Chemical Society

determine blood flow velocity and glucose concentration.32 Combining quantitative phase microscopy for morphology with the chemical selectivity of Raman imaged the distribution of hemozoin in malaria infected red blood cells.33 The combination of atomic force microscopy with conventional Raman was used to study bacteria co-cultures of mycobacterium and gram-negative bacteria,34 as well as examining damage to cells from chemical exposure.35 The narrow features of Raman bands also suggest improved multiplex-imaging markers. Raman based contrast agents are seeing increasing use. Chemical functional groups that have readily identifiable Raman bands, such as nitriles and alkynes, have been used to provide chemical contrast.36-38 In Figure 2, attaching an alkyne group to a nucleic acid, provides a distinctive Raman marker that can be readily observed without interference from more common functional groups found in cells and tissue.37 A key advantage to these tags is the significantly reduced size, and thus reduced perturbation, associated with attaching them to molecules of interest. Similarly, stable isotope labeling Raman microscopy can provide distinct Raman bands to image processes like the metabolism of lipids in droplets.39 Figure 3 shows how deuterated lipid uptake can be monitored by ratioing the isotope label to other molecular vibrations observed in cells.39 Isotopes, thus provide a distinct Raman shift, but are chemically identical to the natural molecule.


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The use of particles with large Raman cross-sections has also been utilized for biomedical microscopy. Silica particles embedded with carotene were used as resonance Raman tags for cellular imaging.40 Nanodiamond uptake was monitored in a series of carcinoma cells and associated with clathrin dependent endocytosis.41 Carbon nanotubes (CNTs) uptake has also been monitored.42 Bovine serum albumin functionalization was reported to preferentially localize CNTs intracellularly.43 An attractive feature of CNTs is they can be detected and used for theranostic purposes using a handheld Raman spectrometer. 44 Graphene has also been studied as a contrast agent, and surface functionalization was reported to be important to mediate toxicity.45 Interpretation of the immense data associated with Raman imaging has resulted in increased use of multivariate analysis methods. Multivariate analysis was used to classify uterine tissue for cancer diagnosis 46 as well as in vitro diagnosis of glaucoma.47 Analysis of TiO2 and FeO(OH) uptake in lung epithelial cells classified a significant fraction of nanoparticles residing in the cell nuclei.48 PCA analysis of cells treated with different doses of doxorubicin showed dose dependent response associated with changes in fatty acids could be characterized.49 Multivariate loadings were shown to be effective for reproducibly removing background signals using a singular value decomposition method in complex biochemical samples.50 Spectral cross-correlation was reported as an improved method over PCA and K-means clustering approaches for determining subcellular distributions of nanoparticles.51 An over-constrained library based fitting method was reported with advantages for predicting bone fracture.52 Interestingly, a web based tool (WHIDE) developed for mass spectrometry data was suggested for use with Raman to optimize the analysis of the multivariate data produced.53


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Figure 3. THP-1 macrophages were incubated with serum-complexed d31-palmitic acid. (A) The Raman spectra from lipid droplets taken at different incubation times. The inset (B) focuses on the spectral region between 1350-1800 cm-1, which shows increased intensity associated with triglyceride storage. (C) depicts composite Raman images from THP-1 macrophages over time associated with increased fatty-acid uptake. The red represents d31-palmitic acid, which is readily distinguished from the unenriched cellular protein shown in blue. The CD/CH ratio represents amount of deuterated fatty acid molecules in respect to protein composition. Adapted and reproduced from C. Matthaus, C. Krafft, B. Dietzek, B. R. Brehm, S. Lorkowski, J. Popp, Anal. Chem. 2012, 84. 8549-8556. © 2012 American Chemical Society

2. Surface Enhanced Methodologies The ability to obtain spontaneous Raman signals from biological samples has advanced tremendously, but the intrinsically low Raman cross-section requires enhancement to the detection of low concentration analytes. When the excitation laser frequency and the emitted Raman photons are resonant with the localized surface plasmon resonance of a nanostructure, the Raman signal can be enhanced by as much as 1011.54 The increase in Raman signal associated with typically gold and silver nanostructures has improved the sensitivity of Raman for biomedical applications.55 In general, two complementary strategies have been pursued. The first is to make SERS tags, where the distinct spectrum of a Raman reporter molecule enhanced by the nanoparticle provides the detected signal. The second approach utilizes the nanoparticle enhancement to detect the intrinsic biomolecules in the sample. This second approach is similar the rapidly developing technique, tip-enhanced Raman scattering (TERS). All of these approaches have produced impressive results.


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2a. SERS Reporter Tags The ability to increase the unique Raman signal of a molecule bound to a gold or silver nanoparticle(s) provides, bar code like signals that can be used to track molecules similar to fluorescent probes and quantum dots.56 Indeed, these SERS reporters have seen increasing use for cancer detection and other biomedical applications.57 Optimization of probe design has enabled imaging and tracking in diverse problems.56 Properly optimized SERS reporter molecules, such as dye molecules that have both electronic resonance with the excitation laser and surface enhancement, have shown better sensitivity than fluorescence.58 Linking nanoparticles in such a way as to optimize the enhancement in the gap between nanoparticles provided SERS reporters with a linear concentration dependence down to femtomolar concentrations.59 Using a rigid spacer provided an optimized hotspot for the reporter molecules that was used for Raman mapping in glioblastoma cells.60 The use of SERS active nanoparticle clusters of dimers and trimers functionalized with p53 and p63 antibodies enabled Immuno-SERS imaging of prostate tissue with acquisitions of 30 ms/pixel.61 Optimization of hollow gold nanospheres has also been reported for biomedical imaging.62 Different shaped nanoparticles, which can have localized surface plasmon resonances in the near infrared (NIR), have also been reported. Gold nanostars were functionalized to bind to p63 to image prostate tissue.63 Nanostars excited with NIR light can be used for intracellular mapping.64 Gold nanorods with reporter molecules have also been used as NIR SERS reporters.65 Stabilization of individual NIR SERS reporters to avoid aggregation can enable quantitative bioimaging.66 Encapsulation and coating of the SERS active nanoparticles can improve their stability and biocompatibility. Polymers have been reported to improve biocompatibility.67 Encapsulating the nanoparticle and reporter molecule in a silica shell improves the function of SERS tags.68 Chitosan coated silver nanotriangles were used for imaging of lung cancer cells.69 Silver nanoparticles functionalized with nile blue and plant derived compounds were reported to have anti-microbial properties.70 Mercaptohexanoic acid was shown to reduce biotoxicity compared to CTAB in HeLa cells.71 Bovine serum albumin functionalized SERS tags are also reported to have improved biocompatibility.72 Multifunctional SERS tags are also being investigated. Gold core nanoparticles capped with iron oxide give good SERS signals, but also can be manipulated with magnetic fields. 73, 74 Polymer-gold hybrid particles have been suggested as imaging agents that can double as drug-delivery vehicles.75 Dual use tags for Raman and fluorescence have been proposed, where SERS signals have noted advantages for multiplexing.76 SERS reporter molecules utility is often associated with the reporter spectrum that is enhanced by the plasmon resonance. Single wall CNTs with gold and silver nanoparticles were shown to enable rapid imaging in cancer cells.77 Graphene oxide with silver nanoparticles were functionalized to bind to folate receptors common in cancer.78 Molecules such as diethylthiatricarbocyanine iodide (DTTC),79 and molecules with alkyne or olefin moieties were reported as effective SERS reporters.80 Nile red coated silver nanoparticles were used to image C. elegans. 81 The NIR dyes Cy7LA and Cy7.5LA functionalized to gold nanorods were shown to target cancer, but also show evidence of clearance from the liver.82 Porphoryn-lipid conjugates evince a reporter spectrum and also stabilize gold nanoparticles for SERS based imaging.83


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Advances in instrumentation are enabling new uses of SERS tags in vitro and in vivo. One study used radio-labeled gold particles to validate the use of SERS particles detected by an endoscope used for mouse colonoscopies.84 Further work indicates the SERS endoscope is a valid approach for detecting SERS tags in tissue.85 Tunable filters are enabling wide field, multiplexed imaging of SERS reporters.86 Filter based methods are being implemented that take advantage of narrow Raman peaks to remove autoflourescence in wide field imaging.87 Depth profiling from SERS tags 20 mm deep in tissue was reported by surface enhanced spatially offset Raman spectroscopy.88 The 3D reconstruction of multiple SERS nanotags in cells was demonstrated.89 Figure 4 shows how the distinctive signature of different SERS tags could be identified and located in three dimensions.89 3D mapping provides improved correlations between specific biomarkers, which will be important for understanding changes in healthy and diseased cells. Computational models are being developed to test the reliability of SERS reporter molecules for biomedical applications.90 Fitting algorithms are shown to minimize error to less than 2% for identifying SERS nanoparticles for imaging.91

Figure 4. (a) The z-slice of nanotags in Chinese hamster ovarian cells at +0.00 is shown. 4-mercaptopyridine (MPY), 5’5-dithiobis(2-nitrobenzoic acid) (DTNB), and 4-nitrobenzenethiol (NBT) labeled nanotags are shown in blue, green, and red, respectively with (b,c) enlarged regions of nanotag clusters. In 2D, clusters appear to colocalize; however in (d) 3D SERS z-slices for the individual nanotags show spatial distribution. (e-g) Raman spectra from the false color clusters from top to bottom show peaks indicative of all four SERS tags. Reprinted with permission from S. McAughtrie, K. Lau, K. Faulds, D. Graham, Chem. Sci. 2013, 4. 35663572. Published by The Royal Society of Chemistry.


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2b. Intrinsic Signal Enhancements By enhancing the Raman spectra of intrinsic biomolecules in close proximity, SERS enhancements are providing insight into physiological phenomena. Changes in the Raman spectra from endocytosed nanoparticles suggest insight into cellular pathways.92 Gold nanorods, also used for photoacoustic imaging, provided SERS signals that could delineate between normal and tumor boundaries in tissue.93 Silver nanoparticles mixed with normal and diabetic rat pancreatic tissue showed Raman bands indicative of DNA, RNA, proteins, and lipids.94 Differences in DNA/RNA and the proteins transcribed were evident in SERS spectra from nuclear targeted gold nanoparticles in a neuroblastoma cell line.95 Polymer encapsulating nitrogen bubbles with embedded silver nanoparticles were used as an ultrasound image contrast agent; interestingly, SERS from the embedded nanoparticles could identify biomolecules interacting with the microbubbles.96 Aggregation is known to affect, often increase, the SERS signals, and thus controlling the formation of SERS active nanostructures has also been investigated.97 Gold nanoparticles bound to a drug were shown to aggregate upon drug release and give SERS from biomolecules within cells.98 It has been shown that gold nanoparticles can be synthesized within cells for SERS detection. 99, 100 Nanoparticles produced by reduction of gold intracellularly were able to detect chemical signatures for Cr(III) and Cr(IV) within

Figure 5. (A) Diagram of instrumental setup with optics for slit-scanning Raman microscope capable of dark-field microscopy and focused detection of tracked nanoparticles. The laser tracking detection scheme is shown in (B). (C) shows Raman spectra obtained from gold nanoparticles identified in the dark-field images. (D) shows SERS spectra from gold nanoparticles in a macrophage cell at times 0, 120, 240, and 360 seconds with (left) dark-field and (middle) SERS images with (right) corresponding Raman spectra. Scale bar: 5 µm. Adapted and reprinted with permission from A. F. Palonpon, J. Ando, H. Yamakoshi, K. Dodo, M. Sodeoka, S. Kawata, K. Fujita, Nature Protocols 2013, 8. 677-692. © 2013 Nature America, Inc.


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cells. Similarly chromate decorated gold nanoparticles are taken up by cells and can provide Raman signals to monitor chromate reduction.101 A microscope was reported that combined slit scanning for rapid Raman mapping and beam steering to rapidly track and obtain SERS spectra from biomolecules near gold nanoparticles, thus representative of the biological microenvironment in cells.102 Figure 5 shows how the combination of nanoparticle imaging and fast Raman acquisition can be combined in a single instrument.102 By using line mapping, where the excitation is focused to a line and each point on the line spectroscopically resolved, a cell can be mapped in minutes. Incorporating laser tracking enables nanoparticles to be monitored in 2D, and their spectrum obtained rapidly associated their position with biomolecule composition. The particle tracking provides a dark-field image of the cell simultaneously. The combination of these Raman measurements represents a powerful approach for Raman microscopy, capitalizing on signal enhancements in SERS. Other interesting reports have taken advantage of SERS enhancements to monitor the location of biomolecules. One report used the signals generated from nanoparticles with stochastic image reconstruction to generate superresolution imaging. 20 nm spatial resolution was reported from peptide fibrils from cardiomyocytes.103 In another report, nanoparticles grown on graphene oxide enabled detection of cellular components in cancer cells.104 Nanodot arrays functionalized with RGD peptides showed enhancements from cells grown on top.105 Proteins deposited on SERS substrates could be imaged and identified.106 Antibody functionalized gold nanoparticles bound to cell membranes were coated in silver to map membrane components.107 2c. Tip-enhanced Raman Imaging The past decade has seen tremendous progress in the controlled movement of nanostructures at the apex of a scan-probe microscope tip, a technique known as tipenhanced Raman scattering (TERS).108, 109 Most impressively, TERS was demonstrated to image components of individual molecules based on their vibrational modes.110 While experimentally challenging,111 112 there are an increasing number of biological applications. Proteins have both been increasingly characterized by TERS. Images of protein nanotapes were obtained using gap-mode TERS, where Raman bands in the fingerprint region enabled chemical characterization.113 The structure and composition of insulin 114, 115 amyloid,116 and collagen 117 fibrils have also been studied. There is some debate in the literature about why certain bands are sporadically observed in SERS and TERS, in particular the amide-I vibration associated with protein secondary structure.118, 119 Studies indicate that the electric field gradient near the tip, or another enhancing nanoparticle, can selectively probe part of a protein, as demonstrated with biotin and streptavidin ligandreceptor system.120, 121 TERS has been suggested as a route to DNA sequencing, where the change in the Raman spectrum, distinct for each base, is associated with sub-nanometer movement of the TERS tip to read out nucleotides.122 TERS has also been used to investigate more complicated biological systems. The detection of lipid domains has been reported in model bilayers,123 cultured human cells,124 and in bacteria.125 Nanoparticles targeting antigens on the plasma membrane of cells were successfully detected by TERS, suggesting a route to increased sensitivity and selectivity in


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Figure 6. In (A) the dark field image identifies where nanoparticles have bound to surface antigens on the cell membrane. (B) The TERS image can be acquired from regions of the cell membrane identified in the dark-field image. (C) The Raman spectra from the identified particles can be measured and differences in the Raman bands observed provide information about the biomolecules present and enhancements associated the nanoparticle clustering. Adapted and reproduced from K. D. Alexander, Z. D. Schultz, Anal. Chem. 2012, 84. 7408-7414. © 2012 American Chemical Society

intact membranes.126 Figure 6 shows an SW480 cancer cell that was incubated with antibody-fucntionalized gold nanoparticles.126 The increased scattering observed when the nanoparticle probe interacts with a TERS tip suggests a method to simultaneously identify proteins of interest and obtain Raman scattering characteristics of the interaction. Micrometer dimensioned metal tips (μ-TERS) were also reported as a way to selectively enhance residues in the cell membrane.127 Other systems that have been investigated with TERS include studying how the protein osteopontin adsorbs to kidney stones,128 nanooxidation sites on hemoglobin crystals,129 and hemozoin crystals associated with malaria-infection from sectioned erythrocytes.130 Improvements in TERS tips and instrumentation have facilitated use for biomedical applications. One challenge in TERS is discriminating the near-field from the far-field background. By synchronizing detection with the tapping mode frequency, it is possible to remove the far-field contribution.131 Advances in AFM tips appropriate for biological samples will further facilitate biomedical TERS.132 Silver tips are reported to give the largest enhancements, and a photochemical method for attaching silver to AFM cantilevers in situ has been reported.133

3. Nonlinear Methods The resurgence of nonlinear Raman techniques was driven by the development of solid-state laser systems and laser scanning microscopes in the 1990’s. Contrary to spontaneous Raman microscopy with a continuous wave laser, nonlinear Raman methods generally use two near-infrared pulsed lasers to probe the third order polarizability of a given a molecule. As a result of the coherent nature, nonlinear Raman methods yield 103106 increased signal compared to spontaneous Raman measurements. Coherent antiStokes Raman scattering (CARS) is a widespread biomedical tool with chemical sensitivity and video-rate imaging comparable to fluorescence microscopy without the use of exogenous agents.134-136 The CARS platform also allows for a multimodal approach to acquire second harmonic generation (SHG) and two-photon excited fluorescence (TPEF)


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measurements simultaneously, gaining more chemical and morphological information from biological samples.135-138 One of the limitations of CARS microscopy is the nonresonant background inherent with the direct measurement of the third order molecular susceptibility of the vibrational bands in a given sample.139 Analogous to CARS excitation, stimulated Raman scattering (SRS) measures the energy loss and gain in the incident lasers when tuned to a molecular vibration, circumventing the difficulties associated with the nonresonant background while providing video-rate, high resolution images.140-142 3a. Coherent Anti-Stokes Raman Scattering Microscopy In CARS, a pump field (ω , Stokes field (ω , and probe field (ω ; typically ω ω ) interact with a sample in a four wave mixing process. When the pump and Stokes fields are spatially and temporally overlapped with a frequency difference resonant with a molecular vibration, ω ω Ω (beat frequency), coherently driving the molecular oscillators in the focal volume, a strong anti-Stokes signal is generated at ω 2ω ω . The change in the third-order polarizability, P α χ E E E∗ , results in a signal nonlinearly dependent on the incident fields and quadratically dependent on analyte concentration, I α I I .135, 139 In addition, a nonresonant background is generated that shows complex interference with the resonant signal. There have been advances in instrumentation and data analysis to suppress the nonresonant background and reconstruct the spontaneous Raman spectrum from the CARS signal.143-146 The different laser beams necessary for CARS microscopy have been employed in three general ways: two independently tunable pulsed lasers with electronic synchronization, single laser with optical parametric oscillator (OPO) or amplifier (OPA) conversion for tunable signal and idler beams for the pump and Stokes, and fiber based lasers with limited wavelength range.135, 147 A femtosecond pulsed laser or continuum generation for the Stokes beam allows for broadband CARS, exciting multiple molecular vibrations simultaneously.135, 147-149 However, multiplex CARS is restricted in laser power, resulting in poor optical contrast and longer acquisition times. Recently, hyperspectral CARS techniques have been implemented, in which single-band CARS acquisitions are taken sequentially by manually tuning the Stokes beam,150, 151 providing more chemical information from the surrounding biomolecules in the focal volume without sacrificing image quality. Hyperspectral data processing addresses the asymmetry in a CARS measurement and reconstructs the Raman spectra from multiplex CARS images.152-158 However, single-band CARS tuned to 2845 cm-1 is the most common method of nonlinear Raman imaging due to the high concentration of CH2 oscillators in lipids found in biological systems. Single-band CARS imaging is a promising diagnostic tool for cancer and other diseases. Gao et al. were first to differentiate normal and cancerous prostate glandular structures and identify cavernous nerves by ex vivo imaging of CH2 symmetric vibrations at 2845 cm-1 within lesions and utilizing an interactive two-dimensional segmentation algorithm to delineate nuclei boundaries.159 Figure 7 shows a simple iterative clustering algorithm implemented to construct 3D volume partitions from z-stacked CARS images with a user interface to select the cellular nuclei.8, 160 The area of Voronoi Tessellation


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provide nuclear size and length of major and minor axis of the nucleus, along with minimum, maximum, and average distance of neighboring cells based on the Delaunay Triangulation.160 CARS can provide information comparable to traditional histopathological examinations without the use of traditional hematoxylin and eosin (H&E) stains, illustrated in Figure 7 (D-I). Cross-referencing these disease-related distinctions to known histopathological classifications allow for quantifiable identification of various cell types of non-small cell lung carcinomas with 99.56% and 97.78% accuracy of true positives for adenocarcinoma and squamous cell carcinoma, respectively.8, 160-162 Similarly, breast cancer subtypes and grades of carcinoma such as invasive ductal carcinoma and lobular carcinoma were distinguishable from each other with high precision.163, 164 The ability to image biopsy samples with high chemical sensitivity and record characteristic changes for each stage of cancer may exploit the patterns of cellular distortion to better the chances of early diagnosis. More importantly, a pathologist may take up to twenty minutes to inform the surgeon of cancer margins during an intraoperative procedure, whereas CARS imaging and segmented classification can be accomplished quicker.


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Figure 7. (A) shows the instrument schematic and classification strategy. The CARS image is from a small cell carcinoma imaged at 2845 cm-1 demonstrating the segmentation analysis by (B) Voronoi Tessellation and (C) Delaunay Traiangulation. The CARS images and corresponding H&E stain from (D,E) normal lung tissue, (F,G) adenocarcinoma, (H,I) squamous cell carcinoma from the same mouse are shown. Adapted and reprinted with permission from L. Gao, A. A. Hammoudi, F. Li, M. J. Thrall, P. T. Cagle, Y. Chen, J. Yang, X. Xia, Y. Fan, Y. Massoud, Z. Wang, S. T. C. Wong, Differential diagnosis of lunch carcinoma with threedimensional quantitative molecular vibrational imaging J. Biomed. Opt. 2012, 17, 066017, and L. Gao, F. Li, M. J. Thrall, Y. Yang, J. Xing, A. A. Hammoudi, H. Zhao, Y. Massoud, P. T. Cagle, Y. Fan, K. K. Wong, Z. Wang, S. T. Wong, On-the-spot lung cancer differential diagnosis by label-free, molecular vibrational imaging and knowledge-based classification J Biomed Opt 2011, 16. 096004. © 2011, 2012 Society of Photo-Optical Instrumentation Engineers

CARS imaging of lipid distributions has also been exploited for the progression of liver disease. Studies have been done to monitor the transformation of lipid droplets and collagen fibers during the progression of liver steatosis and fibrosis induced in a bile duct ligation rat model over a six week period.165 Differential rates of formation of high lipid droplet accumulation up to the fourth week and an increase of collagen fibers up to the sixth week correlate to severe hepatocyte necrosis.165 Exploring this further, Le at al. promoted hepatotoxicity in transgenic and wild-type mice by feeding them fenofibrate, a drug to impair mitochondrial function, resulting in microvesicular steatosis.166 CARS images of liver tissue revealed that lipid rich nonparenchymal cells of hepatocytes of mild and severe cases of steatosis were detectable. In comparison, standard histopathological examinations with Oil Red O were only able to distinguish severe cases of steatosis. CARS studies have investigated treated and untreated human hairs, providing insight to the composition and changes of protein secondary structures within the hair.167


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Broadband CARS measurements were also used to identify the composition of solid pharmaceuticals.168 A label-free flow cytometry technique was developed using multiplex CARS, which was capable of distinguishing subpopulations of yeast, demonstrating the potential of a stand-alone flow cytometer device. 169 CARS imaging is often complemented by linear vibrational spectroscopies. The in vitro analysis of plaque deposition associated with atherosclerosis progression was further investigated with single-band CARS, infrared (IR) microscopy and a Raman fiber probe.170, 171 Applying complimentary techniques allowed for the Raman and IR identification of cholesterol ester, trimyristin, calcium, phosphate, and collagen. Evaluating the coronal artery and aorta biopsies identified two forms of depositions: cholesterol esters throughout the intima, showing the most abundant lipids, and crystalline calcium localized on the tissue edges.170 Although confocal Raman microscopy obtained the most chemical information, Raman imaging of the same atherosclerotic tissue section took several hours, whereas NLO modalities were achieved in minutes. To better integrate spontaneous Raman with CARS studies, a Raman probe designed with one multimode excitation waveguide centered by 12 multimode collection fibers acquired Raman spectra for 10 seconds at specified locations in a living rabbit’s aorta.171 Afterwards, the rabbits were sacrificed to analyze the plaque deposition by in vitro multimodal CARS imaging in 3D stacks of tissue in two minutes. The spontaneous Raman measurements had little interference from blood, and CARS images provided supplementary information regarding the structural changes of the vessel membranes in normal tissue with high signal CH stretches at 2930 cm-1 versus plaque depositions that show maximum signal at 2850 cm1.171 The combination of a Raman probe analysis prior to CARS imaging allows for in vivo applications in small mammal models to better understand disease development. Multimodal nonlinear optical microscopy (NLOM) is an attractive method for investigating biological tissues. Second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) are nonlinear modalities that occur implicitly within the CARS excitation scheme and provide additional insight. SHG is an energy conserving process (ω 2ω ) sensitive to anisotropic biological structures, which enables the direct imaging of collagen fibers and cholesterol.137, 172 Whereas TPEF experiences an energy loss (ω ! 2ω ) during the excitation of endogenous fluorophores, such as elastin fibers, keratins, flavins, and NAD(P)H.137, 172 These signals can easily be separated with dichroic mirrors, appropriate filters, and multiple detector channels. The resulting overlaid images elucidate the chemical composition and morphology of the biological specimen. Multimodal imaging with PCA brought insight into the dysfunction of meibomian gland associated with evaporative eye disease.151 Single-band CARS measurements were taken sequentially from 2800 to 3050 cm-1, in which CARS and SHG images were obtained while Raman microspectroscopy measurements were recorded at points of interest in the meibomian gland. Manually scanning the beat frequency between frames prolonged the acquisition time, such that a 512x512x50 µm spectral stack was completed in 30 minutes. Variation in lipid fluidity between the acinus and the central duct was shown to affect the health of the meibomian gland.151 The role of cholesterol crystals in plaques of atherosclerotic mice was investigated in a similar fashion.150 In this case, hyperspectral CARS and PCA successfully discriminated lipophilic compounds in a quantitative manner within 20 minutes. This technique was capable of identifying lipid-rich macrophages and


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distinguishing needle and platelet shaped cholesterol crystals within the atherosclerotic lesions.150 The invasion and metastases mechanisms of tumors have been investigated with CARS to improve early diagnosis. Integrating multimodal CARS with Raman microspectroscopy provided the chemical composition of biomolecules in the nonlinear optical images. The lipid mobilization kinetics in metastatic circulating tumor cells in prostate cancer were studied by simultaneously acquiring CARS, SHG, and TPEF images at 10 seconds per frame in a 3D stacking of 20 frames to compose a full volume of the tumor section.173 A significant increase in lipid concentration with slow lipid mobility was observed in metastatic cells, suggesting a biomarker for metastatic cancer.173 A compound Raman microscope for label-free imaging of live cell membranes in a 3D culture was used to evaluate breast tissue polarity.174 The CARS images illustrated the lipid distribution within apical and basal membranes and quantified the percentage of human mammary acini that lost apical polarity induced by the arachidonic acid, a known risk factor in breast cancer. An ex vivo co-culture model system of adipose-tumor epithelial cells that replicates the in vivo mammary environment in breast cancer was also studied with CARS and TPEF imaging.175 Multiplex CARS using a broadband continuum successfully imaged fresh mouse brain tissue.155 Analysis of the CARS images was accomplished with the maximum entropy method and a global fitting algorithm with PCA.152-154, 156-158 The chemoselective hyperspectral imaging distinguished granule cells, surrounding tissues, and single layer of Purkinje cells in the mouse brain within four minutes.155 Studies of pig brain and human brain tumor tissue with multimodal CARS provided classification of tissue types and further evaluation of detected biomolecules in the CH2 and CH3 stretching region. 176, 177 An alternative segmentation analysis (than previously described in Figure 7) was developed to identify nuclei in images of squamous cell carcinoma metastasis from a human brain sample. The segmentation analysis is done in three steps: nuclei localization by gray-scale minima detection, radial search for nuclear boundaries, and verification of segmented nuclei by comparing the optical contrast of CARS and TPEF images.178 This allowed for the statistical analysis of average nuclear size and nucleus-to-cytoplasm ratio, nuclei and cell fractions, and number of nuclei per area, which all contribute to the classification of tissue malignancy. The versatility of NLO modalities have also been implemented on head and neck squamous carcinoma cells.179 Chemical and morphological information provided by CARS, TPEF, and SHG superseded that of H&E stains. However, these accurate diagnostic tools are slow to reach clinical settings. Most recently, a robust, portable fiber based multimodal microscope was developed to facilitate NLOM in an intraoperative setting.180, 181 For proof of concept, a human artery was fixed onto a CaF2 slide, and hyperspectral CARS was achieved by scanning 2700 cm-1 to 3000 cm-1 in 13 cm-1 increments with an average of 2 seconds per image to retrieve a spectrum of the CH stretching region, as shown in Figure 8 (A-D).181 The ratio of CARS intensity between the CH2 stretches at 2849 cm-1 and CH3 stretches at 2930 cm-1 allows for the distinction between regions of lipid and proteins, respectively, seen in Figure 8 (J). The addition of TPEF and SHG signals allow for complete differentiation between triglycerides, lipid droplets, myelin, collagen, elastin fibers, and cellular cytoplasm within an atherosclerotic lesion of a rabbit model.181 This robust microscope is free of maintenance and adjustments, making it more desirable and user


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friendly. This novel instrumentation bypasses the complications of a conventional CARS set-up advancing the applications of nonlinear Raman imaging into a clinical environment. The detection of lipids in multimodal CARS has extensively been used to investigate myelination of the nervous system182-184 Myelin membranes are about 70% lipids and surround the axons in the nervous system and provide electrical insulation for signal conduction. Demyelination is a leading cause in central nervous system disorders such as spinal cord injuries and multiple sclerosis. Imitola et al. coupled CARS with reflectance confocal microscopy for rapid, ex vivo imaging of myelin, axons, and microglia in live mouse tissue with chronic experimental autoimmune ancephalomyelitis (EAE).184 Belanger et al. attached a commercial microendoscope to a clamp underneath the objective of the multimodal CARS microscope to direct the beams into a live mouse for in vivo video-rate imaging of the spinal cord.182 Additional work was done by Galli et al. to assess spinal cord injuries in mice using Fourier Transform IR spectroscopy, confocal Raman, and NLO modalities.185 The lipid distribution describing the invasion of peripheral macrophages and microglia activation was monitored after axon demylination, inflammation, and scarring of the spinal cord injuries.185 Along the same methods of detection, multimodal CARS has also been carried out in the investigation of smooth muscle cells in bioengineered tissue scaffold and neuronal cell growth on polymeric scaffolds,186, 187 the differentiation of stem cells and their derivatives,188, 189 and the differentiation of immune and metabolic cells in tissue.190

Figure 8. (A) CARS and SHG image of human perivascular tissue indicating (1,black) lipid droplets, (2,green) collagen fibers (blue-CARS, cyan-SHG), (3,red) cytoplasm and cell membranes, (4,blue) and myelin sheaths with (B) corresponding CARS spectra. Red square is nonresonant background from CaF2 slide used to normalize spectra shown in (C). (D) Tissue section stained with EVG. (E) NLOM image of an atherosclerotic lesion of a rabbit model composed of individual (F) high-concentrated lipids by CARS, (G), fluorescent lipids and elastin by TPEF, and (H) collagen and cholesterol crystals by SHG, (I) CARS image of CH2 (green) and CH3 (blue) stretches enables the distinction between lipid and protein contribution by a (J) frequency scatter plot, identifying the (1) lipid in plaque, and high-intensity protein in the (2) tunica media and (3) tunica externa. (K) SHG of collagen within the tunica externa and cholesterol in the plaque. Adapted and reproduced from T. Meyer, M. Chemnitz, M. Baumgartl, T. 17 ACS Paragon Plus Environment Gottschall, T. Pascher, C. Matthaus, B. F. M. Romeike, B. R. Brehm, J. Limpert, A. Tunnermann, M. Schmitt, B. Dietzek, J. Popp, Anal. Chem. 2013, 85. 6703-6715. © 2013 American Chemical Society


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Working towards understanding genetics and developmental biology, CARS was used to image all the internal organs of a Drosophila larva.191 Towards a more clinical setting, current NLO techniques have been employed for skin diagnostics.192, 193 A hybrid laser tomograph was reported for in vivo optical biopsies of human skin.193 This instrument can distinguish patients with normal skin and patients with Psoriasis. A variety of skin samples and treatments were also demonstrated to show distinctions between tissue architecture, cell morphology, and exterior applications of sunscreen nanoparticles, cosmetics, and moisturizers.193 The differences in healthy and disease-affected human skin were determined by CARS tomography based on the distribution of intercellular lipids in the stratum corneum.192 Drug delivery has also been investigated. The uptake of polymeric nanomedicines for drug delivery has been reported by CARS, where deuterated polymeric micelles were monitored ex vivo following oral and intravenous dosing into the small intestines, liver, and brain.194 Rago et al. also studied uptake of gold (Au) nanoparticles and iron oxide MRI agents in living cells.195, 196 CARS microscopy is coupled with transmission electron microscopy (TEM) for in vivo detection of single nanoparticles in human healthy epidermal keratinocytes and squamous carcinoma cells.195 Although the CARS microscope is tuned to 2845 cm-1 to image the surrounding lipids, poly(ethylene glycol)-coated Au nanoparticles (NPs) generated a multiphoton induced luminescence signal as a result of interacting with the incident beams. The nonspecific uptake of PEG-Au NPs was not prevalent in the squamous carcinoma cells, as there were very few isolated Au NPs detected, but was prevalent in the healthy keratinocytes.195 Rago et al. also observed the uptake of micrometer-sized iron oxide (MPIOs) MRI contrast agents in human hepatoma cells.196 Differences in CARS responses of the MPIOs and lipids enabled distinction of an MPIO that had been successfully internalized versus localized on the surface of the cell.196 These findings indicate that CARS microscopy can provide insight to inorganic nanostructure interactions in biological systems for improving drug delivery and understanding its effects in the body. The size and temperamental nature of pulsed laser systems combined with intricate optics required for CARS microscopy has impeded use in clinical settings. To overcome these impediments, various fiber-based lasers have been built with fiber optical parametric oscillators,197 tunable fiber oscillating parametric amplifiers,147, 198-201 and a master oscillator power amplifier pump laser.202, 203 An all-spliced laser source was constructed to generate a pump and Stokes beam directly into the microscope for an alignment-free system capable of CARS, SHG, and TPEF measurements.204 Most recently, a compact portable laser scanning microscope, as previously mentioned, is equipped with a multicolor fiber lasers and minimal optics, which can be transported and reassembled in several hours and is competitive for in vitro multimodal imaging with frame sizes ranging from 128x128 to 4096x4096 µm and pixel dwell times of 1 to 256 µs.180 The desire for in vivo CARS imaging has led to development of a fiber based CARS endoscope. Work has been done to demonstrate the beam quality, temporal and spectral characteristics, and overall imaging performance of custom photonic crystal fibers,205 and fiber bundles with polarization control to suppress the four mixing effects within the fiber itself.206-208 Saar et al. designed a piezo scanning fiber with gradient index lenses to detect the CARS and SRS signal of lipids and proteins in mouse skin, shown in Figure 9.209 Smith et al. built a portable, multimodal CARS exoscope with a micro-electromechanical system


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scanning mirror and miniaturized optics capable of CARS, SHG, and TPEF epi-detection.210 The challenges still remain to develop fiber-based systems with efficient delivery of pulsed incident lasers, collection of epi-CARS signal, suppression of four wave mixing effects, and conversion of a rapid scanning mechanism in a robust, cost-effective manner.

Figure 9. (A,B) Schematic of CRS scanning fiber endoscope. (C) Spectra of mouse skin, lipid, and protein. SRS images of (D) sebaceous gland and (E) subcutaneous fat beneath the skin. Simultaneously acquired SRS and CARS images of hairs near the surface: (F, H) proteins and (G, I) lipids. All scale bars: 10 µm. Adapted and reprinted with permission from B. G. Saar, R. S. Johnston, C. W. Freudiger, X. S. Xie, E. J. Seibel, Opt. Lett. 2011, 36. 2396-2398. © 2011 Optical Society of America

3c. Stimulated Raman Scattering Microscopy In stimulated Raman spectroscopy (SRS), a pump field (ω and Stokes field (ω interact with a sample, analogous to stimulated emission. When the frequency difference (ω ω matches a molecular vibration, the pump field experiences a loss in energy (ΔI , stimulated Raman loss, SRL) and the Stokes field experiences a gain in energy (ΔI , stimulated Raman gain, SRG).136, 140 The measured intensities, ΔI α χ I I and ΔI α χ I I , have a linear response to concentration similar to spontaneous Raman signals.140 In comparison, SRS detects the excited vibrational population, whereas CARS detects the vibrational coherence.136 SRS also does not suffer from the nonresonant background issues in CARS. Accordingly, SRS is a desirable analytical tool that provides high resolution images comparable to CARS and chemical information equivalent to spontaneous Raman. SRS is generally performed with two synchronized picosecond pulsed lasers for the pump and Stokes beam with a high-frequency phase-sensitive detection to retrieve the


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small SRL and SRG signal that is generated on the incident laser intensity. A lock-in amplifier is typically used to measure the stimulated Raman loss/gain.136, 140, 142 However, the narrowband incident beams limit the chemical specificity to distinguish overlapping Raman bands. Recent advances in instrumentation are geared towards improving these drawbacks and simplifying the detection scheme for biomedical applications. Freudiger and coworkers utilized a broadband pump with a spatial light modulator based pulse shaper and a narrowband Stokes beam for a spectrally tailored excitation.211 The first spectrum is collected with spectral components in resonance with the target species and a second spectrum with components in resonance with the interfering species. The difference between the two spectra isolates the SRS signal of the targeted species while eliminating the residual signal from the interfering species. This signal subtraction was demonstrated in real-time, in vivo imaging of oleic acid, stearic acid, and proteins in live nematode, C. elegans.211 Several multicolor SRS techniques have been constructed for multiplex measurements.212, 213 Lu et al. devised an SRS microscope with a grating based pulse shaper for excitation, grating based spectrograph for detection, and strip scanning method for increased imaging speed as compared to traditional 2D laser scanning.213 Parallel detector channels allow for the simultaneous collection of SRS signal for multiple frequencies, while linear combination analysis allows for the quantitative extraction of multiple species, such as proteins, lipids, and blood. Alternatively, a different multicolor SRS system was designed with a rapidly tunable picosecond OPO, electro-optical filter, and a line-by-line wavelength tuning mechanism to prevent spectral artifacts.212 Additionally, Ozeki et al. utilized a resonant galvanometer scanner and high-speed lock-in amplifier for frame-byframe tunability and rapid tissue imaging.214 More recently, a continuous wave laser was demonstrated as a low-cost option for SRS by imaging the lipid droplets in fatty liver.215 For cw-SRS, two laser diodes were used for excitation and a resonant amplifier in conjunction with a lock-in amplifier were used for detection due to lower peak powers. Additionally, a dual modulation mechanism was used to remove the electronic background.215 Multiplex SRS and hyperspectral SRS techniques have been implemented to alleviate the restrictions of chemical specificity with narrowband excitation sources.216-219 Fu et al. applied an acousto-tunable filter device that divided a broadband femotosecond laser to a number of wavelength bands that can each be modulated simultaneously.219 Fu and coworkers further developed a chirped pulse femtosecond laser for spectral focusing.216 By converting the optical delay to Raman wavenumbers with a calibration set of known solutions, hyperspectral SRS has a spectral range from 500 to 2000 cm-1.216 When the individual components are known, a nonnegative least-squares algorithm can extract the Raman signal and quantitative chemical composition of a mixture of biomolecules. Another method for hyperspectral SRS has been achieved with an intrapulse spectral scanner to sequentially stack images for each frequency.218 Figure 10 shows that by providing a spontaneous Raman spectrum of known components, multivariate curve resolution analysis is able to quantify the concentration of individual species, such as lipids, proteins, and water in the sample with a total image acquisition time of four minutes.218 These developments are increasing the speed and chemical specificity for biomedical imaging.


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Figure 10. Hyperspectral stacking and multivariate curve analysis allowed for SRS imaging of (a) breast cancer MCF7 cells with (b) spectra retrieval and (P-R) image reconstruction of lipids (red), protein/nucleotide (green), and water (blue), respectively. Adapted and reproduced from D. L. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, J. X. Cheng, Anal. Chem. 2013, 85. 98-106. © 2013 American Chemical Society

Individual cellular components have been imaged by SRS. Zhang and coworkers designed a femtosecond stimulated Raman loss method capable of detecting higher signal than conventional picosecond SRS excitation schemes.220 With high sensitivity, nucleic acids were imaged from high DNA concentrations in Drosophila melanogaster salivary gland cells and changes in composition during cell division.221 In a similar fashion, Dou et al. monitored the intracellular motion of lipid droplets in Drosophila embryos.222 Wang et al. were able to identify fat storage regulatory genes by choosing specific RNAi clones to target specific receptor genes and hormone receptors in the model nematode and image the effects of hindered lipid growth throughout the body.223 More recently, Wei et al. studied newly synthesized proteins by coupling SRS imaging with deuterium-labeled amino acids in various cell lines.224 On a much larger scale, SRS imaging has been used to investigate the chemical composition and morphology of notochord in cephalochordate amphioxus and zebrafish as model animals.225 More widely used as a diagnostic tool, stimulated Raman scattering in combination with coherent anti-Stokes Raman scattering, is known as coherent Raman scattering (CRS) microscopy. Freudiger et al. demonstrated CRS techniques for a multicolored stain free histopathology approach.226, 227 To verify the reliability of CRS, tissues from the brain, heart, kidney, liver, lunch, muscle, ovary, spleen, and top and bottom layer of skin were all imaged by CARS and SRS with the ability to differentiate CH2 and CH3 stretches and hemoglobin. Mice models for breast cancer metastasis, focal brain ischemia, and relapsing-


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remitting EAE for demyelination were imaged by SRS and shown to obtain more cellular details than traditional H&E stains.226 Multicolor SRS was used to detect and classify brain tumor progression, specifically glioma ex vivo in human GBM xenograft mice, based on lipid and protein distributions.227 In vivo epi-SRS brain tumor imaging of the cortical surface was achieved through a cranial window, and exploited tumor infiltration present in SRS images that were not discernible under bright-field microscopy conditions.227 Further investigation of the cortical vasculature around the blood brain barrier was done by Moger and coworkers by coupling SRS imaging with two-photon photothermal lensing microscopy.228 In addition to the CARS studies of atherosclerotic plaques, hyperspectral SRS and SHG have also been used to characterize cholesterol crystals.229 Hyperspectral SRS analysis was able to differentiate between cholesterol monohydrate crystals and other condensed matter, whereas SHG signals confirmed previous reports that differentiated plate and needle shaped plaques. 229 Saar et al. observed that ketoprofen and ibuprofen had different penetration rates and pathways into mouse skin,230 agreeing with previous CARS studies. 3d. Enhancement Mechanisms for Nonlinear Raman Methods The signal enhancements associated with SERS and TERS suggest a powerful method when combined nonlinear Raman methods. This methodology has the potential for increased chemical selectivity and specificity to bring insight into chemical interactions and molecular orientation based on the enhanced spectral features. Additionally, CARS or SRS images may facilitate the location of molecular interactions. Several proof of concept experiments have reported an enhanced CARS signal in solution when silver colloids are present.231-233 Stuewe et al. fabricated a nanovoid Au surface template and observed signals with ~105 higher intensity than bulk CARS signal of benzenethiol.234 Schlucker et al. reported the detection of Au/Ag nanoshells with SERS-labeled p63 antibodies in prostate tissues.235 Frontiera et al. were first to report surface enhanced femtosecond SRS (SE-FSRS) with the fabrication of Au nanoantennas with embedded reporter molecules and comparing SERS and SE-FSRS spectra of trans-1,2-bis(4-pyridyl)ethylene (BPE) functionalized nanoatennas.236 More work has been done with plasmon resonance enhanced four wave mixing microscopy. In this technique, the plasmon modes in metal structures can be enhanced and detected from resonance with the incident beams.237-239 Garrett et al. investigated the uptake of Au nanoshells in live cells.237 CARS imaging of lipids was collected in forward direction, whereas the four wave mixing signal arising from the Au nanoshells was collected in epi-direction. The Au nanoshells generated signal orders of magnitude higher than resonant CARS signal from lipids.237 Similarly, the coherent anti-Stokes emission from Au nanorods was imaged on a silicon wafer substrate.238 Polarization dependent studies of Au nanorod aggregates and single Au nanorods compared to SEM images demonstrated the potential to use Au nanorods as sensitive probes for imaging applications.238 The most recent study in enhancement mechanisms was done by Lin et al., utilizing a radially polarized tip-enhanced near-field CARS (RP-TE-CARS) microscope to image mitochondria isolated from HeLa cells at nanoscale resolution.240 In this set-up, the CARS excitation scheme is incorporated into an AFM with a gold tip with a 10 nm radius. Image quality was evaluated by comparing polystyrene bead images generated from linearly polarized TE-


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CARS and radially polarized TE-CARS with corresponding AFM topography images. RP-TECARS imaged the lipid bilayers of the mitochondrial membrane at 2845 cm-1.240 This imaging platform suggests the potential to investigate biomedical systems at the molecular level. 4. Outlook for the Future. Advances in instrumentation and methodology suggest Raman microscopy is a powerful approach for investigating biomedical applications. Since the initial demonstration of Raman in a microscope, the development of spontaneous Raman, surface enhancement, and nonlinear methodologies have made impressive progress. The intrinsic vibrational modes of molecules provide chemical specificity and multiplex chemical detection in a non-destructive approach. It is expected that further advances in instrumentation and methodology will continue in the coming years. In the last couple years, there seems to be increasing use of Raman microscopy with other techniques, where the information gained from tandem measurements provides additional insight relative to performing the same measurements sequentially. Multimodal nonlinear imaging is one example. Tandem, AFM-Raman and Tip-enhanced Raman microscopy are another. The complementary information can provide new insights that overcome traditional limitations, such as the diffraction limit for spatial resolution. Increased understanding of enhancements associated with metal nanostructures has also enabled impressive studies. Raman spectroscopy that can compete with fluorescence in an applied manner suggests new approaches to ultrasensitive optical microscopy. It will be interesting to see if nonlinear methods can further capitalize on these enhancement mechanisms. The complexity of an enhancement mechanism generated from multiple excitation beams and interfering signal generated from the metallic susceptibility further complicates the parameters for these types of experiments. Additional work is needed to establish a reliable and reproducible approach to generate enhanced CARS or SRS signal and to implement these methodologies for biomedical research. Raman continues to make impressive strides and ongoing improvements in all areas of instrumentation and methodology should translate the capabilities of Raman microscopy from “bench top to bedside” in clinical research and mainstream medicine. 5. Biographies Karen A. Antonio received her B.S. in Chemistry from California State University, San Bernardino in 2011. She is currently a Ph.D student in the Department of Chemistry and Biochemistry at the University of Notre Dame. Her dissertation research focuses on utilizing nonlinear Raman techniques to monitor chemical interactions in cellular systems. Zachary D. Schultz received his B.S in Chemistry from the Ohio State University in 2000 and a Ph.D. in Chemistry from the University of Illinois at Urbana-Champaign in 2005. He did postdoctoral research at the National Institute of Standards and Technology from 2005-2007 and in the Laboratory of Chemical Physics at the National Institute of Diabetes and Digestive and Kidney Diseases from 2007-2009. In 2009 he began his current position


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as an assistant professor in the Department of Chemistry and Biochemistry at the University of Notre Dame. He also holds a concurrent faculty appointment in the Department of Chemical and Biological Engineering at Notre Dame. His current research focuses on the development of Raman instrumentation using plasmonic enhancements for chemical imaging and ultrasensitive detection in biomedical systems. 6. Acknowledgement. The authors are grateful for support from the University of Notre Dame and a Cottrell Scholar Award from Research Corporation for Science Advancement to ZDS. KAA acknowledges support from the National Science Foundation Graduate Research Fellowship Program Grant DGE-1313583. 7. References. 1. J. L. Abraham, E. S. Etz, Science 1979, 206. 716-718. 2. Y. Harada, T. Takamatsu, Curr. Pharm. Biotechnol. 2013, 14. 133-140. 3. M. Schmitt, B. Dietzek, U. Neugebauer, C. Krafft, P. Roesch, J. Popp, Endoskopie Heute 2012, 25. 262-267. 4. M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, K. Fujita, Proceedings of the National Academy of Sciences of the United States of America 2012, 109. 28-32. 5. M. J. Nakamura, K. Hotta, K. Oka, Plos One 2013, 8. e71739. 6. Z. D. Schultz, Australian Journal of Chemistry 2011, 64. 611-616. 7. K. Majzner, A. Kaczor, N. Kachamakova-Trojanowska, A. Fedorowicz, S. Chlopicki, M. Baranska, Analyst 2013, 138. 603-610. 8. L. Gao, A. A. Hammoudi, F. Li, M. J. Thrall, P. T. Cagle, Y. Chen, J. Yang, X. Xia, Y. Fan, Y. Massoud, Z. Wang, S. T. C. Wong, J Biomed Opt 2012, 17. 066017. 9. M. Ashtikar, C. Matthaus, M. Schmitt, C. Krafft, A. Fahr, J. Popp, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 2013, 50. 601-8. 10. L. Franzen, C. Mathes, S. Hansen, M. Windbergs, J Biomed Opt 2013, 18. 061210. 11. C. Matthaus, S. Schubert, M. Schmitt, C. Krafft, B. Dietzek, U. S. Schubert, J. Popp, ChemPhysChem 2013, 14. 155-161. 12. T. Chernenko, R. R. Sawant, M. Miljkovic, L. Quintero, M. Diem, V. Torchilin, Molecular Pharmaceutics 2012, 9. 930-936. 13. G. Romero, Y. Qiu, R. A. Murray, S. E. Moya, Macromolecular Bioscience 2013, 13. 234241. 14. J. Baik, G. R. Rosania, Molecular Pharmaceutics 2011, 8. 1742-1749. 15. M. A. Calin, S. V. Parasca, R. Savastru, M. R. Calin, S. Dontu, J. Cancer Res. Clin. Oncol. 2013, 139. 1083-1104. 16. H. Lui, J. Zhao, D. McLean, H. Zeng, Cancer Research 2012, 72. 2491-2500. 17. H. G. Schulze, S. O. Konorov, J. M. Piret, M. W. Blades, R. F. B. Turner, Analyst 2013, 138. 3416-3423. 18. C. Krafft, B. Belay, N. Bergner, B. F. M. Romeike, R. Reichart, R. Kalff, J. Popp, Analyst 2012, 137. 5533-5537.


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Biochemical Info

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Raman Shift

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