Stimulated Raman versus Inverse Raman: Investigating Depletion Mechanisms for Super-Resolution Raman Microscopy

Super-resolution fluorescence microscopy has been critical in elucidating the nanoscale structure of biological systems. However, fluorescent labels bring difficulties such as perturbative labeling steps and photobleaching. Thus, label-free super-resolution techniques are of great interest, like our group's 2016 stimulated Raman scattering (SRS) technique, stimulated Raman depletion microscopy (SRDM). Inspired by stimulated emission depletion microscopy, SRDM uses a toroidally shaped beam to deplete the signal formed on the edges of the focal spot, resulting in SRS signal being detected from only a subdiffraction limited region. In initial works, the cause of the depletion was not thoroughly characterized. Here, we conclusively demonstrate suppression mechanisms in SRDM, while also contrasting approaches to super-resolution Raman microscopy on the Stokes and anti-Stokes sides of the spectrum. By monitoring the depletion of both the SRS and inverse Raman scattering (IRS) signal at a range of depletion powers, we observed other four-wave coherent Raman pathways that correspond to the introduction of the femtosecond depletion beam. In addition, we showed the depletion of the IRS signal, paving the way for a super resolution imaging technique based on IRS, inverse raman depletion microscopy (IRDM). Combined, SRDM and IRDM offer label free super-resolution imaging over a large spectral range to accommodate a variety of different sample constraints.

Automated summary

Super-resolution fluorescence microscopy plays a crucial role in understanding the nanoscale structure of biological systems, but the use of fluorescent labels brings challenges. Therefore, label-free super-resolution techniques like stimulated Raman scattering (SRS) and stimulated Raman depletion microscopy (SRDM) have attracted great interest. This study demonstrates suppression mechanisms in SRDM and explores super-resolution Raman microscopy on different sides of the spectrum. The findings lead to the development of inverse raman depletion microscopy (IRDM) and offer label-free super-resolution imaging over a wide spectral range.