Li Xuesong, Lam Wen Jiun, Cao Zhe, Hao Yan, Sun Qiqi, He Sicong, Mak Ho Yi, Qu Jianan Y
Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Biophotonics Research Laboratory, Clear Water Bay, Kowloon, Hong Kong SAR, China.
Hong Kong University of Science and Technology, Division of Life Science, Clear Water Bay, Kowloon, Hong Kong SAR, China.
J Biomed Opt. 2015 Nov;20(11):110501. doi: 10.1117/1.JBO.20.11.110501.
The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.
本研究的主要目标是证明受激拉曼散射（SRS）作为一种新的成像方式能够被整合到飞秒（fs）非线性光学（NLO）显微镜系统中。高脉冲峰值功率的飞秒光源通常用于多模态非线性显微镜，以实现对多个NLO信号的高效激发。然而，在飞秒激发下，亚细胞脂质和囊泡结构的SRS成像分别由于光谱分辨率差和缺乏化学特异性而受到蛋白质的显著干扰。我们开发了一种独特的飞秒激发NLO显微镜，能够快速采集SRS和多个双光子激发荧光（TPEF）信号。在转基因秀丽隐杆线虫动物的体内成像中，我们发现通过基于来自含色氨酸的内源性蛋白质和溶酶体相关细胞器的TPEF信号交叉过滤假阳性脂质信号，成像系统能够将SRS信号高度准确地分配给脂质。此外，我们证明了多模态NLO显微镜系统能够顺序成像脂质结构结构/内容物以及与脂质代谢密切相关的细胞器，如线粒体、溶酶体和内质网。
