Xiong Hanqing, Qian Naixin, Miao Yupeng, Zhao Zhilun, Chen Chen, Min Wei
Department of Chemistry, Columbia University, New York, NY, 10027, USA.
Light Sci Appl. 2021 Apr 20;10(1):87. doi: 10.1038/s41377-021-00518-5.
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.
受超分辨率荧光显微镜的革命性影响启发，超分辨率拉曼成像因其比荧光成像具有更高的化学特异性而长期受到关注。然而，与荧光相比，振动对比度本质上不太敏感，即使在强激光激发功率下，其分辨率提升也仅略高于衍射极限。因此，在光学远场实现生物相容性超分辨率振动成像仍是一项巨大挑战。2019年，受激拉曼激发荧光（SREF）被发现，它是一种超灵敏的振动光谱技术，结合了拉曼散射的高化学特异性和荧光检测的超高灵敏度。在此，我们开发了一种以SREF为对比机制的新型超分辨率振动成像方法。我们首先确定了反斯托克斯荧光背景在阻碍直接采用超分辨率荧光技术方面的不良作用。然后，我们设计了一种调频（FM）策略来去除宽带背景，并实现了高对比度的SREF成像。借助新合成的SREF染料，我们实现了具有纳米光谱分辨率的多色调频SREF成像。最后，通过将受激辐射损耗（STED）与无背景调频SREF相结合，我们完成了具有高对比度的超分辨率振动成像，即STED-FM-SREF，其空间分辨率仅由信噪比决定。在我们的原理验证演示中，在中等激光激发功率下，生物系统中的分辨率提高了两倍多，通过优化仪器和成像探针，这一结果还将进一步优化。凭借其超分辨率、高灵敏度、振动对比度和适度的激光激发功率，STED-FM-SREF显微镜有望助力广泛的应用。
