Pulse-Picking Multimodal Nonlinear Optical Microscopy
Pulse-Picking Multimodal Nonlinear Optical Microscopy
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DOI：
10.1021/acs.analchem.2c03284
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发表时间：
2022-10-25
影响因子：
7.4
通讯作者：
Zhang, Chi
中科院分区：
文献类型：
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作者：
Clark, Matthew G.;Gonzalez, Gil A.;Zhang, Chi
非线性光学显微镜技术可以以无标记的方式绘制生物样品中的化学成分。通常用于成像的非线性光学过程包括多光子激发荧光（MPEF）、二次谐波产生（SHG）和相干拉曼散射（CRS）。飞秒激光器由于需要高峰功率用于激发而通常用于MPEF和SHG，而皮秒激光器由于需要高光谱分辨率而优选用于CRS。因此，将CRS与MPEF和SHG集成用于化学成像是具有挑战性的。我们开发了一种基于声光调制器的脉冲拾取策略，该声光调制器可以对激光脉冲序列的占空比进行编程，从而在低输入平均功率下显著增加脉冲峰值功率。这种方法提供了强增强的非线性光学信号，并使高光谱相干反斯托克斯拉曼散射（汽车）显微镜兼容MPEF和SHG多模成像在低激光平均功率。脉冲拾取方法还能够评估和比较不同平均和峰值功率水平的激光脉冲的光毒性。使用细胞动力学和样品形态学变化评估生物样品的光扰动，从而选择最佳激光功率以获得最佳灵敏度和最小光毒性。
Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.