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<p>Vibrational spectroscopy is a powerful tool for spectral imaging of biological samples, thanks to its narrow bandwidth (10 cm⁻¹) compared to fluorescence. Single-molecule vibrational spectroscopy has been demonstrated with near-field amplification as in surface-enhanced Raman spectroscopy or fluorescence detection as in stimulated Raman excited fluorescence and bond-selective fluorescence-detected infrared-excited spectro-microscopy. However, these methods often require elaborate sample preparation or sometimes generate background signals when unintended processes lead to fluorescence emission. In response to these issues, we developed electronic resonance stimulated Raman scattering (ER-SRS) to achieve single-molecule sensitivity in far-field vibrational microscopy without relying on fluorescence detection. ER-SRS has encountered difficulties due to large electronic backgrounds. To overcome this, we employed Raman-amplified nonfluorescent molecular probe (RANMP) alongside our synchronously pumped, independently tunable double optical parametric oscillators for effective optimization of the signal-to-background ratio. The optimization of probe and light source allowed us to successfully detect ER-SRS signal from single particles in solution and from single molecules embedded in polymer matrix. ER-SRS combined with RANMP provides single-molecule sensitivity without fluorescence detection, enabling applications in biological and chemical imaging, particularly in multiplexed imaging.</p>

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Fluorescence-free single-molecule microscopy via electronic resonance stimulated Raman scattering

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月13日

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  • Sumin Oh,
  • Yunji Eom,
  • Ha Yeon Kim,
  • Ayushi Tripathi,
  • Sungnam Park,
  • Han Young Woo,
  • Sang-Hee Shim

摘要

Vibrational spectroscopy is a powerful tool for spectral imaging of biological samples, thanks to its narrow bandwidth (10 cm⁻¹) compared to fluorescence. Single-molecule vibrational spectroscopy has been demonstrated with near-field amplification as in surface-enhanced Raman spectroscopy or fluorescence detection as in stimulated Raman excited fluorescence and bond-selective fluorescence-detected infrared-excited spectro-microscopy. However, these methods often require elaborate sample preparation or sometimes generate background signals when unintended processes lead to fluorescence emission. In response to these issues, we developed electronic resonance stimulated Raman scattering (ER-SRS) to achieve single-molecule sensitivity in far-field vibrational microscopy without relying on fluorescence detection. ER-SRS has encountered difficulties due to large electronic backgrounds. To overcome this, we employed Raman-amplified nonfluorescent molecular probe (RANMP) alongside our synchronously pumped, independently tunable double optical parametric oscillators for effective optimization of the signal-to-background ratio. The optimization of probe and light source allowed us to successfully detect ER-SRS signal from single particles in solution and from single molecules embedded in polymer matrix. ER-SRS combined with RANMP provides single-molecule sensitivity without fluorescence detection, enabling applications in biological and chemical imaging, particularly in multiplexed imaging.