Abstract
Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.
Highlights
The advent of far-field super-resolution fluorescence microscopy[1,2,3,4] has greatly sharpened our vision in the microscopic world over the past two decades[4,5,6]
The stimulated emission depletion (STED)-FM-Stimulated Raman Excited Fluorescence (SREF) image shows richer textures and sharper edges than that of the FM-SREF image and exhibits spectrally sharp vibrational contrast at the same time (Fig. 5e–h). These results proved STEDFM-SREF as a biocompatible super-resolution vibrational imaging technique
Insufficient sensitivity has long been the bottleneck of vibrational imaging, which largely accounts for the difficulty of pushing vibrational imaging beyond the diffraction limit
Summary
Difficulty of direct coupling of STED with SREF imaging In SREF excitation, a fluorophore is first excited by stimulated Raman excitation to the target vibrational excited state (orange line and red line in Fig. 1a, b), a probe beam (orange line in Fig. 1a, b) is used to further promote the fluorophore from vibrationally excited state. The absorption and emission of the new dye are nearly identical to that of Rh800 (Fig. 3b), and can have the SREF signal of its nitrile mode (whose resonance is shifted by isotope editing) excited with our current laser system This dye cannot be used together with Rh800 for two-color imaging in conventional fluorescence microscopy. E, when excited at the vibrational resonance of the nitrile mode of Rh800, the structure with the smallest full width at half maximum (FWHM) is ~400 nm under FM-SREF, whereas the same structure shows a ~180 nm FMHM under STED-FM-SREF with a modest STED power (6 mW) (Fig. 4g), indicating a more than two times improvement of the resolution. STED-FM-SREF imaging of the same structure with pump wavelength set at the off vibrational resonance of the nitrile mode shows no net signal (Fig. 4f), which again confirmed the backgroundfree property of our FM strategy coupled with the additional STED excitation. These results proved STEDFM-SREF as a biocompatible super-resolution vibrational imaging technique
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