Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy

Department of Chemistry and Chemical Biology, Harvard University, Cambridge (MA).
Nature Photonics (Impact Factor: 32.39). 01/2011; 5(2):103-109. DOI: 10.1038/nphoton.2010.294
Source: PubMed


Label-free microscopy with chemical contrast and high acquisition speed up to video-rate has recently been made possible by stimulated Raman scattering (SRS) microscopy. While SRS imaging offers superb sensitivity, the spectral specificity of the original narrowband implementation is limited, making distinguishing chemical species with overlapping Raman bands difficult. Here we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species based on tailored multiplex excitation of its vibrational spectrum. This is done by spectral modulation of a broadband pump beam at a high-frequency (>1MHz), allowing detection of the stimulated Raman gain signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live C.elegans.

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    • "Yet the logic of the coherent accumulation, which leads to the high selectivity is identical, and the theoretical techniques used for both analytic treatment and numerical simulation of the molecular dynamics are equivalent. The proposed coherent Raman oscillator is inherently different from other common methods of coherent Raman spectroscopy [14] [15], such as Raman fluorescence spectroscopy, stimulated Raman spectroscopy (SRS) [16] [17] and coherent anti-Stokes Raman spectroscopy (CARS) [18] [19] [20] (where just recently important contributions were reported using two frequency combs [21, 22]). All those well-established methods measure molecular vibrational levels in the ground electronic potential, and therefore, tune the pump field away from all absorption bands to ensure a purely virtual Raman transition between ground levels. "
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    ABSTRACT: Free induction decay is the coherent emission of light that follows the excitation of a medium by a short pulse. During the coherence time of the medium ($T_2$), all atoms/molecules oscillate 'in unison', forming a macroscopic dipole that emits light as a large coherent antenna, 'broadcasting' information on the quantum state of the atoms/molecules and its dynamical evolution. We present an optical oscillator, where the coherent dipole emission from a dynamical wave-packet, is amplified beyond the lasing threshold. By placing a molecular medium in an optical cavity that is synchronously pumped by a frequency comb laser, emission from the excitation of one pump pulse can return to the medium with subsequent pump pulses, allowing stimulated amplification. When threshold is crossed, a broadband coherent oscillation is achieved, bearing information on the coherent wave-packet dynamics inside the medium. We analyze theoretically this coherent Raman oscillator and simulate thoroughly it's dynamics under most realistic conditions for a model system of Alkali dimers ($Li_2, K_2$) in a hot gas cell ($100-300^{\circ}C$), showing that the oscillation condition is well within reach. If realized, this coherent Raman oscillator can open avenues for precise measurement of vibrational dynamics in molecules.
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    • "Therefore, it is difficult to specifically detect molecules with overlapping Raman bands. For further improving the specificity, tailored excitation SRS microscopy was developed [12]. This technique uses narrowband Stokes pulses and spectrally tailored pump pulses to detect specific molecules. "
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    ABSTRACT: To date, medical imaging of tissues has largely relied on time-consuming staining processes, and there is a need for rapid, label-free imaging techniques. Stimulated Raman scattering microscopy offers a three-dimensional, real-time imaging capability with chemical specificity. However, it can be difficult to differentiate between several constituents in tissues because their spectral characteristics can overlap. Furthermore, imaging speeds in previous multispectral stimulated Raman scattering imaging techniques were limited. Here, we demonstrate label-free imaging of tissues by 30 frames/s stimulated Raman scattering microscopy with frame-by-frame wavelength tunability. To produce multicolour images showing different constituents, spectral images were processed by modified independent component analysis, which can extract small differences in spectral features. We present various imaging modalities such as two-dimensional spectral imaging of rat liver, two-colour three-dimensional imaging of a vessel in rat liver, spectral imaging of several sections of intestinal villi in mouse, and in vivo spectral imaging of mouse ear skin.
    Nature Photonics 11/2012; 6(12). DOI:10.1038/NPHOTON.2012.263 · 32.39 Impact Factor
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    • "The other corner is occupied by narrowband coherent anti-Stokes Raman scattering (CARS) with just one spectral discriminating line, quadratic concentration dependence and a non-resonant background, but with tremendous speed. In between we fi nd a variety of techniques such as spectrally scanning CARS (Ganikhanov et al. 2006, Kano 2010 ), amplitude shaped stimulated Raman scattering (SRS) (Xie et al. 2011 ) and different incarnations of broadband CARS (Rinia et al. 2007, Motzkus et al. 2009, Parekh et al. 2010 ). In the Optical Sciences Group at the University of Twente we have pursued the use of phase to improve selectivity without sacrifi cing speed in both narrowband and broadband CARS, both of which will be considered in this review. "
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    ABSTRACT: The phase of the molecular response can be exploited to improve selectivity without sacrifi cing speed in both narrow-band and broadband coherent anti-Stokes Raman scattering (CARS) microscopy, both of which will be considered in this review of the work that was performed in our group.
    03/2012; 31(1). DOI:10.1515/revac.2011.122
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