Selective suppression of CARS signal with three-beam competing stimulated Raman scattering processes

Abstract

Coherent Raman scattering spectroscopy and microscopy are useful methods for studying the chemical and biological structures of molecules with Raman-active modes. In particular, coherent anti-Stokes Raman scattering (CARS) microscopy, which is a label-free method capable of imaging structures by displaying the vibrational contrast of the molecules, has been widely used. However, the lack of a technique for switching-off the CARS signal has prevented the development of the super-resolution Raman imaging method. Here, we demonstrate that a selective suppression of the CARS signal is possible by using a three-beam double stimulated Raman scattering (SRS) scheme; the three beams are the pump, Stokes, and depletion lights in order of frequency. Both pump-Stokes and pump-depletion beam pairs can generate SRS processes by tuning their beat frequencies to match two different vibrational modes, then two CARS signals induced by pump-Stokes-pump and pump-depletion-pump interactions can be generated, where the two CARS signals are coupled with each other because they both involve interactions with the common pump beam. Herein, we show that as the intensity of the depletion beam is increased, one can selectively suppress the pump-Stokes-pump CARS signal because the pump-depletion SRS depletes the pump photons. A detailed theoretical description of the coupled differential equations for the three incident fields and the generated CARS signal fields is presented. Taking benzene as a molecular system, we obtained a maximum CARS suppression efficiency of about 97% with our experimental scheme, where the ring breathing mode of the benzene is associated with pump-Stokes-pump CARS, while the C-H stretching mode is associated with the competing pump-depletion SRS process. We anticipate that this selective switching-off scheme will be of use in developing super-resolution label-free CARS microscopy.

Far-Field Super-Resolution Vibrational Spectroscopy

“…For example, Choi et al point out that a depletion power of 2 TW cm –2 is incompatible with biological material. However, less efficient depletion will still allow them to see improvements in resolution . The results and simulations discussed here represent promising steps toward this goal and toward offering alternative microscopy options to fluorescence-based methods.…”

“…Another potential approach to resolution improvement is using a spatially shaped beam to selectively suppress signal generation in a defined area. Recent advances have included both theoretical descriptions − and experimental implementations , of approaches to obtain subdiffraction-limit resolution with Raman microscopy. They all propose the use of a doughnut-shaped beam to suppress Raman signal (Figure e), which is inspired by the fluorescence-based STED method …”

“…The scheme that resulted in the highest signal suppression efficiency (∼97%) involved introducing an SRS process to compete with a CARS process that uses the same pump beam (Figure ). They suggest using a doughnut-shaped beam to drive SRS processes that deplete the CARS signal on the edges of the focal spot, resulting in a smaller PSF central lobe. Numerical simulations from the group found that the PSF of this subdiffraction-limit point will be capable of being narrowed by increasing either the intensity ratio between the Stokes and depletion beams or the Raman gain coefficients, although no imaging experiments have been performed to date …”

“…When the depletion beam energy is 250 nJ, the CARS signal is depleted by 97%. Adapted with permission from ref . Copyright 2018 Royal Society of Chemistry.…”

Stimulated Raman imaging below the diffraction limit with a MHz laser

“…[18,19] To avoid the use of fluorophores, label-free superresolution techniques have been the focus of significant research efforts. [4,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] Techniques based on Raman spectroscopy have drawn interest due to the fact that it probes vibrational modes that are inherent in a given molecule. Even if an analyte of interest has a Raman spectrum that makes it difficult to isolate in a matrix containing similar vibrations, a very small vibrational tag can be used to provide contrast.…”