We propose and implement a far-field spectroscopic system for imaging below the diffraction limit without the need for fluorescence labeling. Our technique combines concepts from Stimulated Emission Depletion (STED) microscopy and Femtosecond Stimulated Raman Spectroscopy (FSRS). The FSRS process generates signal through the creation of vibrational coherences, and here we use a toroidal-shaped decoherence pulse to eliminate vibrational signal from the edges of the focal spot. The nonlinear dependence on decoherence pulse power enables subdiffraction imaging. As in STED, the resolution is in theory infinitely small given infinite decoherence pulse power. Here, we first experimentally demonstrate that the photophysical principles behind our super-resolution Raman imaging method are sound. We then prove that addition of the decoherence pulse significantly improves the spatial resolution of our microscope, achieving values beyond the diffraction limit. We discuss future directions for this technique, including methods to reach resolution on the order of ten nanometers.
important to note that since most of the represented in 1 is proof-of-concept, acquisition times and power values may differ significantly when these techniques are applied to actual systems of interest. 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. 63 The results and simulations discussed here represent promising steps toward this goal and toward offering alternative microscopy options to fluorescence-based methods.
Carborane RAFT agents are introduced as tunable multi-purpose tools acting as 1H NMR spectroscopic handles, Raman probes, and recognition units.
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.
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