Eighth Condensed Phase and Interfacial Molecular Science (CPIMS)

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Towards single molecule stimulated Raman scattering detection Sunney Xie, Gary Holtom, Dan Fu Dept. of Chemistry and Chemical Biology, Harvard University 12 Oxford Street, Cambridge, MA 0213, Cambridge, MA 02138 E-mail: xie@chemistry.harvard.edu Single molecule spectroscopy and imaging has profoundly deepened our understanding of basic chemical and biological processes. Tremendous progress has been made in the last two decades in singlemolecule spectroscopy techniques, especially based on fluorescence detection. The heavy reliance on fluorescence detection for single-molecule spectroscopy is taken for granted by virtue of background free detection. However, most molecules are not naturally fluorescent. Labeling those molecules could be either difficult or affect their function, thus preventing the direct study of those molecules. Therefore, it is important to explore other avenues in single molecule detection and imaging. Recent success of single molecule absorption detection further motivated us to tackle the larger challenge: single molecule Raman detection. Raman scattering is an inelastic light scattering process that reveals chemical information. Well-resolved vibrational bands are sensitive to both electronic and molecular structure changes. We aim to use stimulated Raman scattering spectroscopy to achieve single molecule Raman scattering detection. Previously we have achieved an SRS detection limit of 50 µM for retinyl palmitate with 1 sec integration time. We have employed two different approaches to further improve the sensitivity of our current SRS system. Figure 1: (A) Spontaneous Raman spectra of 1mM β-carotene dissolved in cyclohexane normalized by the solvent peak at 801 cm -1 . The C=H stretching peak is at 1521 cm -1 . (B) SRS on resonance (1521 cm -1 ) and off resonance (1501 cm -1 ) signals from βcarotene at different concentrations. Integration time is 100 ms. The first approach utilizes near-resonance effect to increase the Raman cross-section. We used visible picosecond lasers as both pump and Stokes beam to excite the SRS of the β-carotene molecule, which has strong absorption near 470 nm. Based on spontaneous Raman measurement (Figure 1a), we observed that visible excitation at 532 nm presents an increase in cross-section of more than 400 fold compared to excitation at 785 nm, at the same excitation power. We measured the detection limit with near-resonant SRS using a flow cell. Solutions of β-carotene in ethanol at increasing concentrations were flowed through a home-made flow cell and the SRS signals were recorded with a lock-in amplifier (100ms time constant). Figure 1(b) shows the concentration-dependent SRS signal. A detection limit of 1µM can be achieved. We also noticed a substantial contribution of off-resonance signal, which is very likely coming from two-color two photon absorption. The second approach we have undertaken to improve sensitivity is using chirped femtosecond lasers as excitation sources. Stimulated Raman scattering efficiency depends on a number of parameters other than wavelength, including spectral bandwidth, pulse duration and repetition rate. A common laser source for SRS imaging is a saturable absorbed mode-locked Nd:YVO4 laser which has a pulse duration of 7ps and a synchronous pumped optical parametric oscillator (OPO) with similar output pulse duration. This 215
pulse duration is a few times longer than the vibrational lifetime of typical chemical bonds, which reduces the excitation efficiency. We adopted a different laser system for SRS excitation. The laser system we used is a home-built Yb:KGW femtosecond laser and a synchronously pumped femtosecond OPO (Figure 2a). In order to reduce the excitation bandwidth, we chirped both lasers with appropriate lengths of glass rods to about 2ps in pulse duration. We demonstrated theoretically and experimentally that the excitation efficiency of this laser system is equivalent to picosecond laser systems with the same pulse duration. With shorter pulse duration, we were able to achieve higher sensitivity at the same power level. In addition, this system allows us to incorporate a frequency modulation (FM) scheme that can effectively remove non-Raman contribution (Figure 2b-c). As an example, we have demonstrated that FM-SRS can image DNA directly in mammalian cells at the fingerprint frequency 790 cm -1 . Figure 2: (a) FM-SRS based on chirped femtosecond lasers. The Stokes beam is modulated by an electro-optical modulator and a polarization beamsplitter. The delay of the retroreflecting mirror determines the probed Raman frequency. By changing the relative delay of the two retroreflecting mirrors after the PBS, an effective frequency modulation is applied. (b) SRS image of a mitotic HeLa cell obtained from a single SRS arm probing at 790 cm -1 . (c) FM- SRS image of the same cell with the relative Raman shift separated by 20 cm -1 . (d) DAPI epi-fluorescence image of the same cell shows the same contrast of the condensed chromosomes. In summary, we have already achieved single molecule detection sensitivity for absorption using a pump-probe type imaging technique. SRS spectroscopy and microscopy uses a similar detection scheme and has already achieved 50µM detection sensitivity. We are further pushing the sensitivity limit of SRS detection towards single molecule. In theory, near-resonance enhancement could provide up to 400-fold increase in detection sensitivity for β-carotene. We have already improved it to 1µM. A new laser system based on chirped femtosecond laser excitation could also improve the sensitivity as well as eliminating non-Raman background such as absorption. We believe that with further technical developments, we will ultimately achieve the goal of single molecule Raman detection. Publications of DOE/BES sponsored research 1. Fu, Dan; Holtom, Gary; Freudiger, Christian W.; Xu, Zhang; Xie, X. Sunney “Fast Fast Raman spectral imaging using chirped femtosecond lasers” J. Phys. Chem, submitted (2012). 2. Chong, Shasha; Min, Wei; Xie, X. Sunney "Ground-State Depletion Microscopy: Detection Sensitivity of Single-Molecule Optical Absorption at Room Temperature" J. Phys. Chem. Lett. 1, 3316-3322 (2010). 3. Saar, Brian G.; Zeng, Yining; Freudiger, Christian W.; Liu, Yu-San; Himmel, Michael E.; Xie, X. Sunney; Ding, Shi-You "Label-Free, Real-Time Monitoring of Biomass Processing with Stimulated Raman Scattering Microscopy" Angew. Chem. Int. Ed. 49, 5476-5479 (2010). 4. Min, Wei; Jiang, Liang; Xie, X. Sunney "Complex Kinetics of Fluctuating Enzymes: Phase Diagram Characterization of a Minimal Kinetic Scheme" Chem. Asian J. 5, 1129-1138 (2010). 5. Lu, Sijia; Min, Wei; Chong, Shasha; Holtom, Gary R.; Xie, X. Sunney "Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy" Appl. Phys. Lett., 96, 113701 (2010). 216
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Eighth Condensed Phase and Interfac
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Potential energy landscape of the (
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Agenda
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Session III Chair: Jay A. LaVerne,
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Table of Contents
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Transition Metal-Molecular Interact
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Ultrafast Electron Transport across
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CPIMS Principal Investigator Abstra
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VUV LDPI image of a polymer photore
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Publications based on DOE support (
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entirely quenched, presumably due t
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Future Plans In addition to the TiO
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The relative path indexes of the ur
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We have recently carried out molecu
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have been obtained with γ-rays sug
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Two novel plasma devices have been
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(Figure 2) can be adsorbed exclusiv
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3. Future Plans Our planned work de
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Figure 1: Snap shots from molecular
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6. Varilly, P., A. J. Patel and D.
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hypothesis that delocalized charges
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Efforts will be made to observe ele
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Photochemistry and Solvation Dynami
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complementary to work we have carri
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The Co3O4 films for the optical pum
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chamber will allow us to heat the s
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calculations. Additional PMF calcul
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the gas solute first needs to cross
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laser system, which is an infrared
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Publications (10/1/2009-present) fo
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interest are: (i) What is the diffu
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and anilino groups attached to the
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had been formed. This coincides wit
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Figure 1. (A) Simplified bR photocy
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Figure 4. Photocurrent density of b
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single-file diffusion and cannot ca
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PUBLICATIONS SUPPORTED BY USDOE CHE
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arrangement. However, the hydrogen
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(5) “Water Dynamics in Salt Solut
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from high- energy x-ray diffraction
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concentrated aqueous solutions. The
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molecule’s center of mass, or alt
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Moreover, SHG studies by Saykally
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The effective fragment potential (E
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10. D.D. Kemp, J. Rintelman, M.S. G
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temperature dependent, equilibrium
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mimicking real organic photovoltaic
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100 kHz amplifier, SE-FSRS was succ
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photovoltaics will be explored via
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for laser surface reactions to thes
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This is a significant advantage of
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the STM, which might underlie the u
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Future Plans: Figure 3. One and two
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methane molecule and a lattice atom
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eactive than the Ni, experiment sug
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Fig. 1. PE spectra of M3Oy � (M =
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each of the intermediates obtained
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a regime where each solvent molecul
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observables that arise from subtle
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total potentials. One can see from
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Our calculations have shown that ch
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our understanding of structure and
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viscosity, η, are inversely relate
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photodissociation of the metal - NO
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from a Ru 2p3/2 orbital to an orbit
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Integrated O 2 PSD (ML) Integrated
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anisotropy in the structure of the
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XPS studies have been performed on
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Publications with BES support since
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MLCT (metal to ligand charge transf
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molecule-metal interface without a
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observe the predicted effects, whic
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(4*) Lymar, S. V.; Schwarz, H. A. J
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(τ .820 ns), but we have not been
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B. Photodissociation spectroscopy o
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oth BLYP and PBE. Our results have
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Publications from BES support (2010
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understanding of factors that contr
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Our talk will discussion our initia
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Figure 1 shows the band structure o
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likely to also occur in low coordin
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These results are promising indicat
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This page is intentionally left bla
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method recently developed by Prende
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and coordination regions. A differe
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two ions I - [3] and IO3 - [7]. One
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"Probing the Hydration Structure of
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world-wide scientific user base sup
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10) C. Zhang, M.E. Grass, A.H. McDa
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Page 187 and 188: 2. Optical Stark study of PtF (Acce
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Page 197 and 198: approach on a model phenol-amine pr
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Page 211 and 212: gas phase using electrospray ioniza
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Page 225 and 226: Studies of structure and reaction d
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Page 237 and 238: Participant List Dr. Musahid Ahmed
Page 239 and 240: Professor Kenneth Eisenthal Columbi
Page 241 and 242: Dr. Ireneusz Janik Notre Dame Radia
Page 243 and 244: Professor Eric Potma University of
Page 245: Dr. James Wishart Brookhaven Nation
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Eighth Condensed Phase and Interfacial Molecular Science (CPIMS)

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