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

Abstract

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.

Quantifying Bacteriorhodopsin Activity as a Function of its Local Environment with a Raman-Based Assay

“…We believe that this difference in response to pH may be due to the presence of a pH gradient across the cellular membrane or differences in the membrane structure leading to changes in bR’s efficiency. In order to determine how the local environment of the cellular membrane affects the activity of bR, we hope to combine our bR activity assay with super-resolution Raman imaging methods in order to image the activity of bR across a patch of purple membrane. − These measurements will help to inform us on how the local membrane environment can affect the function of the proteins found within it.…”

SERS microscopy as a tool for comprehensive biochemical characterization in complex samples

“…Examples include the definition of sampling volumes in single living cells for spontaneous Raman scattering, 1 or the widespread utilization of stimulated Raman processes, in particular coherent anti-Stokes Raman scattering (CARS) 2 and stimulated Raman scattering (SRS), 3 as well as the more recent development of super-resolution Raman microscopy approaches. 4,5 Confining a sampling volume to the near field of a scanning probe in atomic force microscopy or scanning tunneling microscopy in tipenhanced Raman scattering (TERS) 6 has even revealed vibrations of individual molecular bonds. 7 In surface-enhanced Raman scattering (SERS), the excitation of localized surface plasmon resonances (LSPR) in nanostructures leads to the generation of high local optical fields in the proximity of the nanostructures, and thereby to the enhancement of the excitation field and the scattering field, by a respective frequency-dependent field enhancement factor A(n).…”

“…Examples include the definition of sampling volumes in single living cells for spontaneous Raman scattering, 1 or the widespread utilization of stimulated Raman processes, in particular coherent anti-Stokes Raman scattering (CARS) 2 and stimulated Raman scattering (SRS), 3 as well as the more recent development of super-resolution Raman microscopy approaches. 4,5 Confining a sampling volume to the near field of a scanning probe in atomic force microscopy or scanning tunneling microscopy in tip-enhanced Raman scattering (TERS) 6 has even revealed vibrations of individual molecular bonds. 7…”