Stimulated Raman imaging below the diffraction limit with a MHz laser

Abstract

Super‐resolution techniques based on Raman spectroscopy could be implemented as label‐free alternatives to fluorescence‐based techniques due to their chemically specific signal and multiplexing potential. In previous work, we developed a stimulated Raman‐based imaging technique that surpassed the diffraction limit using a toroidally shaped pulse to deplete the signal in a spatially defined area. The photophysical principles of depletion and improved spatial resolution were demonstrated using a 1‐kHz laser with high peak power that were able to efficiently drive depletion. However, this laser was not well suited for soft matter samples, which degraded under the intense beams. To improve the biological capabilities of our setup, we have adapted our technique for a 2.04‐MHz laser system. The increased repetition rate produces far more spectra per second, allowing us to decrease the pulse powers while maintaining reasonable acquisition times. Using the 2.04‐MHz laser, we are able to demonstrate strong signal depletion of 62% and resolution enhancements of 52%, which is comparable with the metrics obtained with the 1‐kHz laser. However, further improvements in resolution were not achieved despite increases in the depletion beam energy relative to the other beams. Frequency‐resolved optical gating analysis of the fundamental output of the 2.04 MHz laser indicated an inconsistent pulse phase and duration. We expect that the inconsistent depletion was a result of this pulse profile and conclude that efficient depletion depends on highly reproducible and stable laser pulses.

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.…”

Preface to the special issue dedicated to Professor Richard P. Van Duyne (1945–2019)

“…They expect that the observed inconsistent depletion is a result of the inconsistent pulse profile and conclude that efficient depletion depends on highly reproducible and stable laser pulses. [ 14 ]…”

“…They expect that the observed inconsistent depletion is a result of the inconsistent pulse profile and conclude that efficient depletion depends on highly reproducible and stable laser pulses. [14] Baumberg, Nijs, et al show that highly reproducible SERS spectra can be achieved with relative standard deviations below 1% by carefully controlling the parameters connected to the signal generation, from particle gap sizes to aggregation times. In addition, they show that sources of variance in substrates can be turned into tuning parameters, allowing for control in the sensitivities of the substrates when carefully characterized and controlled.…”

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

“…The initial report of our SRS depletion technique resulted in a resolution improvement of 40%. The technique, which we have termed stimulated Raman depletion microscopy (SRDM), has since been improved to allow for resolution improvements of up to 52% . The nonlinear dependence of the signal depletion and depletion power makes SRDM likely able to achieve greater resolution improvement at lower powers than the other SRS-based methods discussed above.…”