Significance: Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. Aim: We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging. Approach: We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity. Results: First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics. Conclusions: The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.

中文翻译：

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改善三光子显微镜的激光标准

意义：三光子激发显微镜在生物组织中的穿透深度是两光子成像的两倍至三倍，因此有可能彻底改变体内生物过程的可视化。但是，与两光子成像中使用的激光器的即插即用操作和性能不同，三光子显微镜提出了新的技术挑战，需要仔细研究激光脉冲的保真度。目的：我们实施了最先进的脉冲测量，并开发了创新的技术来检查用于三光子显微镜的激光器的性能。然后，我们演示了如何使用这些技术来提供脉冲形状，脉冲能量以及脉冲间强度变化的精确测量，所有这些最终都会影响成像。方法：我们构建了廉价的工具，例如，第二谐波产生频率分辨光学选通（SHG-FROG）装置和深存储器二极管成像（DMDI）装置，用于检查激光脉冲保真度。结果：首先，SHG-FROG显示出非常大的三阶色散（TOD）。这种程度的相位失真会阻止激光脉冲的有效时间压缩达到其理论极限。此外，当使用获得激光脉冲持续时间的常规方法时，例如，当使用自相关器时，TOD不能被量化。最后，DMDI显示了在与三光子成像相关的时标上检测脉冲到脉冲强度波动的有效性，而使用传统仪器和统计数据无法捕获这些波动。结论：由TOD引起的单个激光脉冲的畸变通过防止激光脉冲的有效压缩并降低非线性激发的效率，对三光子成像提出了重大挑战。此外，不应假定可接受的低脉冲间振幅变化性。特别是对于三光子显微镜中使用的低重复频率激光源，脉冲间的差异也会降低图像质量。如果三光子成像将成为主流，激光制造商可能会使用我们的诊断程序来改善系统设计，最终用户可能会使用我们的诊断程序来验证其当前和未来成像系统的性能。特别是对于三光子显微镜中使用的低重复频率激光源，脉冲间的差异也会降低图像质量。如果三光子成像将成为主流，激光制造商可能会使用我们的诊断程序来改善系统设计，最终用户可能会使用我们的诊断程序来验证其当前和未来成像系统的性能。特别是对于三光子显微镜中使用的低重复频率激光源，脉冲间的差异也会降低图像质量。如果三光子成像将成为主流，激光制造商可能会使用我们的诊断程序来改善系统设计，最终用户可能会使用我们的诊断程序来验证其当前和未来成像系统的性能。