Farinella Deano M, Roy Arani, Liu Chao J, Kara Prakash
University of Minnesota, Department of Neuroscience and Center for Magnetic Resonance Research, Minneapolis, Minnesota, United States.
Neurophotonics. 2021 Jan;8(1):015009. doi: 10.1117/1.NPh.8.1.015009. Epub 2021 Mar 6.
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 . 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. 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. 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. 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. 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.
与双光子成像相比，三光子激发显微镜在生物组织中的穿透深度提高了两倍到三倍，因此有可能彻底改变生物过程的可视化方式。然而，与双光子成像中即插即用的激光操作和性能不同，三光子显微镜带来了新的技术挑战，需要更深入地研究激光脉冲的保真度。我们实施了最先进的脉冲测量，并开发了创新技术来检测三光子显微镜中使用的激光性能。然后，我们展示了如何使用这些技术来精确测量脉冲形状、脉冲能量和脉冲间强度变化，所有这些最终都会影响成像。我们构建了廉价的工具，例如二次谐波产生频率分辨光学门控（SHG-FROG）装置和深度记忆二极管成像（DMDI）设备，以检测激光脉冲保真度。首先，SHG-FROG揭示了非常大的三阶色散（TOD）。这种程度的相位失真阻碍了激光脉冲有效地压缩到其理论极限。此外，使用传统的获取激光脉冲持续时间的方法（例如使用自相关仪）时，无法量化TOD。最后，DMDI显示了在与三光子成像相关的时间尺度上检测脉冲间强度波动的有效性，而使用传统仪器和统计方法则无法捕捉到这些波动。由TOD引起的单个激光脉冲失真给三光子成像带来了重大挑战，因为它阻止了激光脉冲的有效压缩并降低了非线性激发的效率。此外，不应假定脉冲间幅度变化可接受地低。特别是对于三光子显微镜中使用的低重复率激光源，脉冲间变化也会降低图像质量。如果三光子成像要成为主流，我们的诊断方法可被激光制造商用于改进系统设计，并被最终用户用于验证其当前和未来成像系统的性能。
