Towards stimulated Raman scattering spectro-microscopy across the entire Raman active region using a multiple-plate continuum.

Stimulated Raman scattering (SRS) has attracted increasing attention in bio-imaging because of the ability toward background-free molecular-specific acquisitions without fluorescence labeling. Nevertheless, the corresponding sensitivity and specificity remain far behind those of fluorescence techniques. Here, we demonstrate SRS spectro-microscopy driven by a multiple-plate continuum (MPC), whose octave-spanning bandwidth (600-1300 nm) and high spectral energy density (∼1 nJ/cm-1) enable spectroscopic interrogation across the entire Raman active region (0-4000 cm-1), SRS imaging of a Drosophila brain, and electronic pre-resonance (EPR) detection of a fluorescent dye. We envision that utilizing MPC light source will substantially enhance the sensitivity and specificity of SRS by implementing EPR mode and spectral multiplexing via accessing three or more coherent wavelengths.

Electronic Preresonance Stimulated Raman Scattering Spectromicroscopy Using Multiple-Plate Continuum.

Stimulated Raman scattering (SRS) spectromicroscopy is a powerful technique that enables label-free detection of chemical bonds with high specificity. However, the low Raman cross section due to typical far-electronic resonance excitation seriously restricts the sensitivity and undermines its application to bio-imaging. To address this bottleneck, the electronic preresonance (EPR) SRS technique has been developed to enhance the Raman signals by shifting the excitation frequency toward the molecular absorption. A fundamental weakness of the previous demonstration is the lack of dual-wavelength tunability, making EPR-SRS only applicable to a limited number of species in the proof-of-concept experiment. Here, we demonstrate the EPR-SRS spectromicroscopy using a multiple-plate continuum (MPC) light source able to examine a single vibration mode with independently adjustable pump and Stokes wavelengths. In our experiments, the C═C vibration mode of Alexa 635 is interrogated by continuously scanning the pump-to-absorption frequency detuning throughout the entire EPR region enabled by MPC. The results exhibit 150-fold SRS signal enhancement and good agreement with the Albrecht A-term preresonance model. Signal enhancement is also observed in EPR-SRS images of the whole Drosophila brain stained with Alexa 635. With the improved sensitivity and potential to implement hyperspectral measurement, we envision that MPC-EPR-SRS spectromicroscopy can bring the Raman techniques closer to a routine in bio-imaging.