Stimulated Raman imaging below the diffraction limit with a MHz laser

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. A super‐resolution imaging technique based on stimulated Raman spectroscopy would allow for fluorophore‐free imaging below the diffraction limit. We adapt a previously demonstrated technique for a higher repetition rate laser in order to improve compatibility with biological materials. We compare proof‐of‐concept results between the two laser systems, including subdiffraction limit resolution and discuss challenges to further resolution improvements.