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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    • "The most popular non-linear Raman techniques are CARS (coherent anti-stokes Raman scattering) and SRS (stimulated Raman scattering). Both approaches are used extensively to make the valuable Raman information accessible for microscopy applications [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]. However, these systems are usually based on expensive short pulse laser sources which require elaborate techniques for hyperspectral recordings and fiber delivery is very challenging due to non-linear effects in fiber -especially self-phase modulation). "
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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.
    Full-text · Article · Mar 2014
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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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