Dr. Fran AdarHORIBA Scientific
Raman Spectroscopy: The Synergism between the Instrumentation Evolution and the Emerging Applications
Dr. John Chalmers and Dr. Fran Adar
The evolution of the instrumentation used to detect the Raman effect, from the initial instrument used by CV Raman himself, through to the current use of multichannel detectors on grating-based spectrographs with microscope sampling devices, will be reviewed, while indicating what fields of science have been explored during these time periods. In the earliest period we will indicate how Raman spectra were originally used as an aid to determine molecular structure. During the 1960's it was successfully used to study the physics of semiconducting materials and devices. The introduction of the microscope in ~1974 as a sampling device first simplified experimental conditions, but also provided information on a scale commensurate with many questions of microstructure and with problems of manufacturing defects. Curiously, while the original concept of the microscope in the early 1970's was focused on Raman imaging, technological limitations prevented its practical implementation. As the ability to acquire high quality map data over a region of interest improved, development of multivariate statistical means of treating the data has provided high quality Raman images that are now yielding solutions to problems.
Prof. Ping-Heng TanInstitute of Semiconductors, Chinese Academy of Sciences, P. R. CHINA
Ultra-Low-Frequency Raman modes in two-dimensional layered materials
The fast progress of graphene research, fuelled by the unique properties of this two dimensional (2D) material, paved the way to experiments on other 2D layered materials (LM). Atoms within each layer in 2D LMs are held together by covalent bonds, while van der Waals interactions keep the layers together. The interlayer van der Waals (vdW) coupling and low frequency phonon modes, and how they evolve with the number of layers, are important for both the mechanical and electrical properties of 2D layered materials.
We will address the recent advance on the experimental micro-Raman technique to access the ultra-low phonon mode in 2D LMs.[2,3] The research progress on the rigid-layer vibrations both for shear and layer breathing modes in various 2D LMs from graphenes to transition metal dichalcogenides are reviewed. Their scaling rule with layer number can be modeled by an atomic linear chain model, with general applicability to any layered material, allowing a reliable diagnostic of their thickness.
We also uncover the shear mode for MLGs and show that it provides a direct measurement of the interlayer coupling. The corresponding shear modes can be well-fitted with a Breit-Wagner-Fano lineshape, which arises as quantum interference between the shear mode and a continuum of Raman-active electronic transitions. This makes it a probe for the quasiparticles near the Dirac point by quantum interference. By measuring the interlayer shear modes[4,5] using Raman spectroscopy, we also probe the coupling at the interface between two artificially stacked few-layer graphenes, rotated with respect to each other.
- F. Bonaccorso, P.H. Tan and A. C. Ferrari, ACS Nano, 7:3 (2013) 1838-1844.
- X. Zhang, P. H. Tan, et al., Phys. Rev. B, 87 (2013) 115413.
- P. H. Tan et al., Nature Materials, 11:4 (2012)294-300.
- P. H. Tan, et al., Phys. Rev. B, 2014, submitted.
- J. B. Wu, P. H. Tan, et al., Nature Communications, 2014, submitted.
Dr. Michael J. PelletierPfizer Global Research and Development
Low-Wavenumber Stokes and Anti-Stokes Raman Microscopy for Pharmaceutical Tablet Characterization
Discrimination between polymorphs of an active pharmaceutical ingredient (API) in a pharmaceutical product is important because an undesired API crystal form may have different bioavailability or stability characteristics than the desired form. Micro-Raman mapping has proven useful for nondestructive, spatially resolved identification of API polymorphs even at low API levels in the drug product.
Raman spectral polymorph discrimination is usually based on small band shifts in the fingerprint region (400-1800 cm-1). These shifts result from functional groups, such as a carbonyl group, experiencing different microenvironments in the different crystal forms. Low-wavenumber Raman bands (-200 to 200 cm-1) result from larger scale motions, such as deformation of the molecular skeleton or even the whole unit cell. Since low-wavenumber Raman bands are more directly related to the entire crystal structure than vibrational bands from small functional groups, they often improve API polymorph discrimination. The anti-Stokes segment of this spectral region provides additional Raman intensity, though no additional spectroscopic information, that can contribute to improved accuracy and spectral artifact detection. Volume holographic filters for laser intensity rejection allow simultaneous acquisition of the Stokes/anti-Stokes low-wavenumber region along with the fingerprint region of Raman spectra.
Using multivariate analysis, we demonstrate and evaluate polymorph identification from combined low-wavenumber and fingerprint Raman spectra of pharmaceutical tablet image pixels. Multivariate discrimination between polymorphs is surprisingly robust to spectral variation due to crystal orientation. The strong and polymorph-selective API bands typical of the low wavenumber Raman spectral region allow polymorph-specific mapping at rates as high as 50 spectra per second.
Michael J. Pelletier1, Shawn Mehrens1, Christine C. Pelletier2
- Pfizer Worldwide R&D
- Gales Ferry, CT
Prof. Lukas NovotnyETH Zürich, Photonics Laboratory, Switzerland
Near-field Raman Microscopy and Spectroscopy of Nanocarbon Materials
We use a laser-irradiated optical antenna to establish a localized optical interaction with a sample surface. A hyperspectral image of the sample surface is recorded by raster-scanning the antenna over the sample surface and acquiring a Raman scattering spectrum pixel-by-pixel. We apply this type of near-field spectroscopy to map out phonons and excitons in nanocarbon materials (graphene, carbon nanotubes) with a spatial resolution of 10nm. The method is able to resolve local defects as well as interactions with the local environment. We observe that defects lead to a band renormalization and to trapping of excitons.
Near-field Raman imaging of single-walled carbon nanotubes. The image has been recorded by raster-scanning the sample underneath a laser-irradiated metal tip (antenna) and integrating, for each image pixel, the photon counts that fall into a narrow spectral bandwidth centered around the G-line at n = 1594 cm?1 (indicated by the yellow stripe in B). (B) Raman scattering spectrum recorded on top of the nanotube, (C) Enhancement of the G-line signal as a function of tip-sample distance.
Prof. Christian Pellerin and Dr. Marie Richard-LacroixDepartment of Chemistry, University of Montreal
Raman Spectroscopy of Individual Electrospun Fibers
Electrospinning is widely used for producing nanofibers that may be used in tissue engineering, selective filtration, etc. However, the control of their properties is limited by the fact that most characterization techniques require bundles of fibers to obtain information about their structure and orientation. We have shown that polarized confocal Raman microscopy is a powerful tool for characterizing individual electrospun nanofibers using poly(ethylene terephthalate) and polystyrene as model systems. Highly reproducible polarized spectra with good signal-to-noise ratio allow quantifying molecular orientation, crystallinity, and conformation.
In conducting this study, we were faced with a limitation of the conventional orientation quantification procedure for polymers: the need to measure the depolarization ratio of an isotropic sample and to assume that it remains fixed in the oriented samples. The error associated with this can be very significant. We have recently proposed a new procedure to quantify orientation of polymers by Raman spectroscopy based on the most probable orientation distribution function. Its concept and practical application will be described.
Dr. Neil EverallIntertek-Wilton, UK
Industrial Problem Solving with Raman Spectroscopy
Raman spectroscopy occupies an interesting position in the gamut of techniques available to the industrial scientist. On the one hand, it may not be the best possible technique for obtaining a particular piece of information when working in ideal circumstances. For example, if one needs to solve the structure of a soluble, volatile molecule, then one would probably turn first to NMR and mass spectrometry, if available. If one needs to image morphology with 1?m resolution, electron microscopy would be the first choice. Fundamental studies of crystalline phases would probably begin with X-ray diffraction, not Raman. However, in the real world, samples present themselves in awkward, non-ideal circumstances. They may be stubbornly insoluble and non-volatile. We might need to image chemistry with high spatial resolution or analyse tiny defects in situ in larger objects. We might want to look inside a reactor in real time, not having the luxury to remove a sample for NMR or mass spec. Perhaps we have to study crystalline domains with a spatial resolution beyond that which is routinely achievable by XRD, or we might want to analyse samples non-destructively, perhaps in-vivo. In all these cases, and more, we can find that Raman offers a better alternative to the "gold standard" technique, and in fact, Raman is often the only viable tool to solve a particular problem.
In this talk, real case histories are drawn from the workload of a lab that supports the R&D, production and technical service activities of a wide range of industries. The examples are chosen to illustrate Raman spectroscopy's unique offering in an industrial environment, focusing particularly on the use of confocal Raman microscopy. This includes Raman imaging of the structures of blends, coatings and laminates, analysis of defects in composites, mapping of cure in UV-cured coatings, and studies of polymer crystallinity on the micron scale. All of these examples would have been difficult or impossible to achieve using other techniques.
Prof. The-Quyen NguyenNorthwestern University
Raman Spectroscopy goes out Helping Patients in the Operating Room
The-Quyen Nguyen,1 Jennifer Giltnane,2 Melinda Sanders,2 Mark Kelley,3 Ginger Holt,4 Anita Mahadevan-Jansen1
1Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232; 2Division of Pathology, 3Division of Surgical Oncology, 4Department of Orthopaedic Surgery and Rehabilitation, Vanderbilt University Medical Center, Nashville, TN 37232
Surgery is the primary treatment for cancers to completely remove the tumor. It has been shown that the presence of cancer cells within the margin of resected tumors is strongly correlated with the risk of local tumor recurrence. If the margin contains tumor cells, patients have to undergo a second surgery to remove more tissue. Margins are thus directly correlated to the success of cancer surgeries. Standard histopathology provides a definitive diagnosis of margin status, but results may take several days. Consequently, there is a need for a rapid, accurate, automated guidance tool that can be used during tumor resection to assure complete removal in a single procedure.
We have developed a 3-dimensional scanning device that can measure the entire surface of a resected tumor. The device automatically reconstructs the 3D image of a tumor, scans its entire surface and evaluates its surgical status in real-time using optical spectroscopy. Tests have been carried out on risk-reducing mastectomy and sarcoma specimens and the outcomes have demonstrated a consistent correlation with histopathology results. With this device, intra-operative analysis of margin can be achieved less than 15 minutes. This instrument would be able to diagnose the margin status of a excised tumor while patient is still in the OR so that if more tissue needs to be removed, it can be done immediately rather than in a second surgery.
The device will help to ensure complete removal of the tumor with clear margins in a single procedure, thus greatly reducing or eliminating the need for re-operation as well as reducing the time, cost, and anxiety associated with repeat surgeries. The device will perform more quickly and accurately than currently available techniques. It will benefit all patients undergoing surgery for the treatment of cancer, speeding up patient recovery and improving quality of life. As the need for re-operation is eliminated, hundreds of millions of dollars per year can be saved by both patients and the health care industry.
Prof. Igor K. LednevUniversity at Albany, State University of New York
Supremacy and Variety of Vibrational Spectroscopy for Probing Amyloid Fibrils: From UV Raman to VCD and TERS
In spite of the key medical importance of amyloid fibrils, the molecular mechanism of fibrillation is not fully understood. At least in part this is because amyloid fibrils are non-crystalline and insoluble, and thus are not amenable to conventional X-ray crystallography and solution NMR, the classical tools of structural biology. Together with our collaborators we have developed and applied novel experimental approaches based on advanced vibrational spectroscopy for characterizing structure and dynamics of amyloid fibril during the last decade. These include deep ultraviolet resonance Raman (DUVRR) spectroscopy, vibrational circular dichroism (VCD) and tip-enhanced Raman spectroscopy (TERS). In addition to hardware, we developed advanced statistical methods for analyzing spectroscopic data including two dimensional correlation spectroscopy (2DCoS). The application of these complimentary methods for amyloid fibril characterization will be discussed.
We established a detail fibrillation mechanism by detecting the structural intermediates at early stages of fibrillation and determining the sequential order of their appearance through 2DCoS analysis of DUVRR data. DUVRR spectroscopy combined with hydrogen-deuterium allowed us for characterizing the fibril core structure for various fibril polymorphs. A new protein folding-aggregation phenomenon, spontaneous refolding of one fibril polymorph to another was discovered. Fibril polymorphs prepared from the same protein under slightly different pH conditions exhibit opposite chirality according to VSD measurements. Overwhelming majority of structural information accumulated so far about amyloid fibrils are limited to its bulk or core properties. However, the fibril surface determines the biological activity and associated toxicity. TERS offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. We utilized TERS for characterizing the secondary structure and amino acid residue composition of the fibril surface. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. The propensity of various amino acids on the fibril surface and specific surface secondary structure elements were evaluated.
Gala Dinner Guest Speaker: Prof. Mildred DresselhausMIT
My Forty-Year Adventure with Raman Spectroscopy, and the Future
Graphene has been known to the science community since the pioneering work of Wallace in 1947. In this talk I share my over-40-year adventure with Raman spectroscopic studies of nanocarbons. In the 1970s and 1980s we studied single and few graphene layers coming from graphite intercalation compounds. From Raman studies of fullerenes in the 1980s and early 1990s, we moved to spectroscopic studies of carbon nanotubes in the 1990s and then back to graphene in the last decade after Geim and Novoselov started the recent explosion of interest in graphene. For the past 40 years there has been a remarkable growth in nanocarbon research and Raman spectroscopy has played a major role in increasing our understanding of the physics and chemistry of nanocarbons during this long period of time. This overview will address the past, present, and look to the future of graphene research and what lies beyond graphene.
Prof. Sanford A. AsherUniversity of Pittsburgh
UV Raman Studies of Protein and Peptide Structure and Folding Studies
UV Raman excitation into the ~200 nm peptide bond electronic transitions enhance peptide bond amide vibrations of the backbone. A particular band (the amide III3) reports on the Ramachandran psi angle and peptide bond hydrogen bonding. This band is Raman scattered independently by each peptide bond with insignificant coupling between adjacent peptide bonds. Isotope editing of a peptide bond (by replacing the Calpha- H with Calpha- D) allows us to determine the frequency of individual peptide bonds within a peptide or protein to yield their psi angles. Consideration of the Boltzmann equilibria allows us to determine the psi angle Gibbs free energy landscape along the psi (un)folding coordinate that connects secondary structure conformations. The psi angle coordinate is the most important reaction coordinate necessary to understand mechanism(s) of protein folding.
We examine the details of peptide folding conformation dynamics with laser T-jumps where the water temperature is elevated by an 1.9 mM IR nsec laser pulse and we monitor the ~200 nm UV Raman spectrum as a function of time. These spectra show the time evolution of conformation. We will discuss the role of salts on stabilizing conformations in solution.
Prof. Igor ChourpaUniversité de Tours François Rabelais, France
SERS and Fluorescence as Analytical Tools to Study Theranostic Nanosystems
Surface-enhanced Raman scattering (SERS) spectroscopy and fluorescence spectroscopy are analytical techniques well-recognised in several scientific domains, from chemistry to biology. This is due to the strength of these techniques, namely high molecular selectifity and sensitivity enabling single-molecule detection. In the present communication, we will describe how the combination or coupling of SERS with fluorescence can increase even more their potential in analytical and diagnostic applications. In particular, we will focus on complementary use of SERS- and fluorescence- based approaches in the pluridisciplinary research on novel biocompatible nanosystems developed for theranosis (therapy and diagnosis) of cancers[1, 2]. The applications presented will concern several aspects: from nanosystem characterization[3, 4] and study of drug loading/release in suspension[4-6], drug delivery in live cancer cells to development of multimodal biomedical imaging concepts.
- Gautier J, Allard-Vannier E, Munnier E, Soucé M, Chourpa I. Recent advances in theranostic nanocarriers of doxorubicin based on iron oxide and gold nanoparticles. J Control Release. 2013 Jul 10;169(1-2):48-61.
- Gautier J, Allard-Vannier E, Hervé-Aubert K, Soucé M, Chourpa I. Design strategies of hybrid metallic nanoparticles for theragnostic applications. Nanotechnology. 2013 Nov 1;24(43):432002.
- Kaaki K, Hervé-Aubert K, Chiper M, Shkilnyy A, Soucé M, Benoit R, Paillard A, Dubois P, Saboungi ML, Chourpa I. Magnetic nanocarriers of doxorubicin coated with poly(ethylene glycol) and folic acid: relation between coating structure, surface properties, colloidal stability, and cancer cell targeting. Langmuir. 2012 Jan 17;28(2):1496-505.
- Chiper M, Hervé Aubert K, Augé A, Fouquenet JF, Soucé M, Chourpa I. Colloidal stability and thermo-responsive properties of iron oxide nanoparticles coated with polymers: advantages of Pluronic® F68-PEG mixture. Nanotechnology. 2013 Oct 4;24(39):395605.
- Gautier J, Munnier E, Douziech-Eyrolles L, Paillard A, Dubois P, Chourpa I. SERS spectroscopic approach to study doxorubicin complexes with Fe(2+) ions and drug release from SPION-based nanocarriers. Analyst. 2013 Nov 12;138(24):7354-61.
- Šimáková P, Gautier J, Procházka M, Hervé-Aubert K, Chourpa I. Polyethylene-glycol-Stabilized Ag Nanoparticles for Surface-Enhanced Raman Scattering Spectroscopy: Ag Surface Accessibility Studied Using Metalation of Free-Base Porphyrins. J. Phys. Chem. C, Article ASAP.DOI: 10.1021/jp5005709. Publication Date (Web): March 17, 2014.
- Chourpa I, Lei FH, Dubois P, Manfait M, Sockalingum GD. Intracellular applications of analytical SERS spectroscopy and multispectral imaging. Chem. Soc. Rev., 2008, 37: 993 - 1000.
Prof. Ji-xin ChengPurdue University
Microsecond Time Scale Spectroscopic Imaging for In Vivo Molecular Analysis
Raman spectroscopic imaging of highly dynamic systems was inhibited by relatively long spectral acquisition time. Recently developed multiplex coherent anti-Stokes Raman scattering (CARS) microscopy reduced the acquisition time to tens of millisecond. The CARS signal is, however, mixed with a pixel-dependent nonresonant background, which makes quantitative analysis difficult. Here, we report a novel spectroscopic imaging scheme based on parallel lock-in free detection of spectrally dispersed stimulated Raman scattering signal using a homebuilt tuned amplifier array. Our method reduced the spectral acquisition time to 30 microseconds per pixel, which is faster than multiplex CARS by three orders of magnitude. Aided by multivariate curve resolution analysis, we have monitored molecular penetration into skin tissue in situ and in real time. Fast spectroscopic imaging opens a new window for in situ analysis of target molecules in highly dynamic environment such as live cells. The reported technique also holds the potential for direct visualization of chemistry that occurs at microsecond time scale.
Prof. Paul ChampionPhysics Department, Northeastern University
Coherent Low-Frequency Vibrational Motion in Proteins and Biomolecules
Recent studies have demonstrated how either static or transient distortions along specific normal modes of the heme group can help to activate coherent motions induced by ultrashort laser pulses. These coherent motions are associated with heme out-of-plane normal modes that would otherwise be forbidden by symmetry. The distortion-induced enhancement mechanism is most relevant for low-frequency modes, which are particularly susceptible to distortion by the protein environment because of their weak force constants. The protein can evidently "tune" or distort the heme to perform a multitude of different tasks and these distortions can be monitored by measuring the low frequency Raman-active vibrational coherences. Results on several different biomolecular systems demonstrate that vibrational coherence spectroscopy is a sensitive probe of thermally accessible and functionally relevant distortions of the active site heme chromophore. The low frequency motions (?? < kBT?300K?200 cm-1) associated with these distortions are able to extract energy from the thermal bath and utilize it for barrier crossing. These low frequency modes are prime candidates to serve as biochemical reaction coordinates and their ability to mix with other delocalized low-frequency modes of the protein, or with binding partners, offers a potential control mechanism.
Prof. Lawrence D. ZieglerBoston University
In Vitro Cellular Activity Probed by SERS: Applications for Diagnostics and Forensics
Surface enhanced Raman spectroscopy (SERS) excited at 785 nm is found to be a sensitive probe of the metabolic products of bacterial and human cells. Cells removed from the human body undergo characteristic in vitro robust biological activity whose detection can be exploited for biomedical, diagnostic and forensic applications. In particular, the degradation products resulting from energy depletion in bacterial cells provides a unique SERS signature that can be both species and strain specific. This methodology is being developed for diagnosing blood and urinary tract infections with antibiotic specificity. 785 nm excited SERS spectra of human blood and red blood cells (RBCs) are due to blood plasma and hemoglobin, respectively and may be exploited for several biomedical purposes including blood aging monitoring and malria detection. The SERS spectrum of stored whole human blood changes dramatically over ~ 24 hours becoming nearly dominated by hypoxanthine, a metabolite of purine degradation, over this period of time due to its release into blood serum from white blood cells. Tumor cells are well-known to exhibit high metabolic rates compared to normal, non-pathogenic cells. Again, characteristic SERS vibrational signatures due to molecules like adenine, hypoxanthine and NADH appear over the course of several hours from single cancer cells. Thus SERS may provide a procedure for in vitro single cell cancer detection as well as fundamental studies of the effects of genetic or proteomic manipulation for cancer therapy efficacy evaluation. Finally, the use of SERS for trace detection and identification of human body fluids such as blood, semen, vaginal fluid and saliva will be described and demonstrates that SERS can serve as a novel methodology for ultrasensitive forensic identification at crime scenes.
Prof. Wei MinColumbia University
Bioorthogonal Nonlinear Vibrational Imaging
Innovations in spectroscopy principles and microscopy technology have significantly impacted modern biology and medicine. Here we will discuss an emerging chemical imaging platform, stimulated Raman scattering (SRS) microscopy, which can enhance the feeble spontaneous Raman transition by virtue of stimulated emission. When coupled with stable isotopes such as deuterium or exogenous chemical moieties such as alkynes, the resulting bioorthogonal nonlinear vibrational imaging is well suited for probing metabolism of living systems in vivo at microscopic level. Physical principle of the underlying optical spectroscopy and emerging biomedical applications such as imaging lipid metabolism, protein synthesis, protein degradation, DNA replication, RNA synthesis, glucose uptake, and drug tracking will be presented.
Prof. Sunney XieHarvard University, Department of Chemistry and Chemical Biology
Label Free Vibrational Imaging for Medicine
Stimulated Raman scattering microscopy is a label-free and noninvasive imaging technique using vibrational spectroscopy as the contrast. Recent advances have allowed significant improvements in sensitivity, selectivity, robustness, cost; opening a wide range of biomedical applications.