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Simultaneous Dual-Color Stimulated Raman Scattering Biomolecular Imaging with Tailored Supercontinuum Generation and

Le Xin1, Zhiwei Huang1,2,3

  • 1Optical Bioimaging Laboratory, Department of Biomedical Engineering, College of Design and Engineering, National University of Singapore, Singapore, 117576, Singapore.

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|June 13, 2025
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Summary

We developed dual-color stimulated Raman scattering (DC-SRS) microscopy for simultaneous imaging of cellular vibrations. This advanced technique reveals metabolic dynamics and compound localization in living cells and tumor spheroids.

Keywords:
broadband Raman imagingphotonic crystal fiber (PCF)stimulated Raman scattering (SRS) microscopysupercontinuum generation

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Area of Science:

  • Biomedical Optics
  • Cellular Imaging
  • Spectroscopy

Background:

  • Stimulated Raman Scattering (SRS) microscopy enables label-free vibrational imaging.
  • Simultaneous imaging of multiple Raman bands is crucial for complex biological samples.
  • Current SRS techniques often face limitations in simultaneous multi-band detection.

Purpose of the Study:

  • To develop a novel dual-color SRS (DC-SRS) microscopy system.
  • To achieve simultaneous Raman imaging in both high-wavenumber and cell-silent spectral regions.
  • To demonstrate the utility of DC-SRS for live cell and tissue analysis.

Main Methods:

  • Utilized supercontinuum generation from a photonic crystal fiber to generate dual pump beams (800 and 850 nm).
  • Employed a phase-shifted scheme on the Stokes beam (1040 nm) for single photodiode detection.
  • Integrated a 2-channel lock-in amplifier for simultaneous signal acquisition.

Main Results:

  • Successfully demonstrated simultaneous SRS imaging of C-H (2935 cm-1) and C-D/C≡C (2110/2216 cm-1) Raman bands.
  • Revealed subcellular metabolic dynamics and precise localization of deuterated/alkyne-tagged compounds in living cells.
  • Showcased rapid quantitative monitoring of cellular metabolism and drug-cell interactions in tumor spheroids.

Conclusions:

  • The developed DC-SRS microscopy enables simultaneous multi-band Raman imaging with high sensitivity.
  • This technique advances high-resolution metabolic functional imaging in biological and biomedical research.
  • DC-SRS holds significant potential for studying cellular metabolism and drug interactions in complex models.