Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy

Abstract

Recently we have reported electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy as a powerful technique for super-multiplex imaging ( Wei, L. ; Nature 2017 , 544 , 465 - 470 ). However, under rigorous electronic resonance, background signal, which mainly originates from pump-probe process, overwhelms the desired vibrational signature of the chromophores. Here we demonstrate electronic resonant stimulated Raman scattering (er-SRS) microspectroscopy and imaging through suppression of electronic background and subsequent retrieval of vibrational peaks. We observed a change of the vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian as the electronic resonance was approached, in agreement with theoretical prediction. In addition, resonant Raman cross sections have been determined after power-dependence study as well as Raman excitation profile calculation. As large as 10 cm of resonance Raman cross section is estimated in er-SRS, which is about 100 times higher than previously reported in epr-SRS. These results of er-SRS microspectroscopy pave the way for the single-molecule Raman detection and ultrasensitive biological imaging.

Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy

“…With coherent Raman scattering techniques, including coherent anti-Stokes Raman scattering (CARS) 23 and stimulated Raman scattering (SRS) 24 microscopy, imaging speed and sensitivity are significantly improved compared to conventional Raman 25,26 . Moreover, electronic pre-resonance SRS further pushes the sensitivity to sub-μM 27–29 . Furthermore, the most recently reported stimulated Raman exited fluorescence (SREF) microscopy, which encodes the Raman feature into the excitation spectrum of fluorescence emission, has accomplished the long-thought-after goal of detecting single-molecule vibration without plasmonic enhancement 30,31 .…”

Review of Stimulated Raman Scattering Microscopy Techniques and Applications in the Biosciences

“…The resonant Raman effect has been known for a long time, in which the excitation wavelength lies within the absorption band of the chromophore to greatly promote the Raman transition. [ 72 ] Since only the vibration modes related to electronic transitions can be enhanced, resonance Raman scattering can provide rich spectral information of the highly selective excited state structure of the chromophore. However, this signal enhancement is accompanied by a significant reduction in chemical specificity.…”

Coherent Raman scattering microscopy: capable solution in search of a larger audience

“…The principle of resonance Raman scattering can be ported to CRS excitation and correspondingly improve the molecular concentration detection limits. [54][55][56] This strategy requires chromophores that exhibit vibrational modes with good couplings to the electronic resonance. Select fluorophores developed for the purpose of fluorescence labeling display excellent enhancement of the vibrational response of up to 10 6 times when the excitation frequencies are tuned near the electronic transition, as demonstrated for stimulated Raman scattering (SRS).…”

“…Select fluorophores developed for the purpose of fluorescence labeling display excellent enhancement of the vibrational response of up to 10 6 times when the excitation frequencies are tuned near the electronic transition, as demonstrated for stimulated Raman scattering (SRS). 55,56 The result is that such fluorescent probes can be detected through the CRS process at sub-μM concentrations. This is significant, because it opens up the possibility to map distinct probes in a multi-label sample, with the number of different probes ultimately limited by the bandwidth of the Raman line rather than by the bandwidth of fluorescence.…”