Electronic Preresonance Stimulated Raman Scattering Spectromicroscopy Using Multiple-Plate Continuum

Area of Science:

• Chemical Physics
• Spectroscopy
• Bio-imaging

Background:

• Stimulated Raman scattering (SRS) spectromicroscopy offers label-free chemical bond detection but suffers from low sensitivity due to far-electronic resonance excitation.
• Existing electronic preresonance (EPR) SRS techniques lack dual-wavelength tunability, limiting their application to specific molecular species.
• Low Raman cross sections hinder the sensitivity and widespread application of SRS in bio-imaging.

Purpose of the Study:

• To develop and demonstrate an enhanced EPR-SRS spectromicroscopy technique with improved sensitivity and dual-wavelength tunability.
• To overcome the limitations of previous EPR-SRS methods by utilizing a novel light source for broader applicability in bio-imaging.
• To investigate the signal enhancement capabilities of the new technique for label-free chemical bond detection.

Main Methods:

• Utilized a multiple-plate continuum (MPC) light source to achieve independently adjustable pump and Stokes wavelengths for EPR-SRS.
• Interrogated the C═C vibration mode of Alexa 635 by scanning pump-to-absorption frequency detuning across the EPR region.
• Applied the developed MPC-EPR-SRS spectromicroscopy to image the brain of *Drosophila* stained with Alexa 635.

Main Results:

• Achieved a 150-fold enhancement in SRS signal, consistent with the Albrecht A-term preresonance model.
• Demonstrated successful signal enhancement in EPR-SRS images of a whole *Drosophila* brain.
• Validated the dual-wavelength tunability of the MPC light source for interrogating specific vibration modes.

Conclusions:

• The developed MPC-EPR-SRS spectromicroscopy significantly enhances sensitivity for label-free chemical detection.
• This technique overcomes previous limitations in wavelength tunability, expanding the scope of EPR-SRS applications.
• MPC-EPR-SRS holds promise for routine use in bio-imaging due to its improved sensitivity and potential for hyperspectral measurements.