Stimulated Raman imaging below the diffraction limit with a MHz laser
Stimulated Raman imaging below the diffraction limit with a MHz laser
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DOI：
10.1002/jrs.5970
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发表时间：
2020-08
影响因子：
2.5
通讯作者：
Christian T. Graefe;David Punihaole;Michael J Lynch;W. R. Silva;R. Frontiera
中科院分区：
文献类型：
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作者：
Christian T. Graefe;David Punihaole;Michael J Lynch;W. R. Silva;R. Frontiera
基于拉曼光谱的超分辨率技术由于其化学特异性信号和多路复用潜力，可以作为基于荧光的技术的无标记替代品来实现。在以前的工作中，我们开发了一种基于受激拉曼的成像技术，该技术使用环形脉冲来耗尽空间限定区域中的信号，从而超过了衍射极限。使用能够有效驱动耗尽的具有高峰功率的1-kHz激光器，证明了耗尽的物理学原理和提高的空间分辨率。然而，这种激光器并不适合软物质样品，它们在强光束下会退化。为了提高我们装置的生物学性能，我们将我们的技术应用于2.04 MHz激光系统。增加的重复率每秒产生更多的光谱，使我们能够降低脉冲功率，同时保持合理的采集时间。使用2.04 MHz激光器，我们能够证明62%的强信号损耗和52%的分辨率增强，这与使用1 kHz激光器获得的指标相当。然而，尽管相对于其他光束，耗尽光束能量增加，但分辨率没有进一步提高。对2.04 MHz激光器基波输出的频率分辨光学选通分析表明，脉冲相位和持续时间不一致。我们预计不一致的耗尽是这种脉冲轮廓的结果，并得出结论，有效的耗尽取决于高度可重复性和稳定的激光脉冲。
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.