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Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2023-03-15 , DOI: 10.1021/acs.jpcb.3c00302 Risa Sasaki 1 , Shogo Toda 1 , Takaiku Sakamoto 2 , Eiji Sakuradani 2 , Shinsuke Shigeto 1
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2023-03-15 , DOI: 10.1021/acs.jpcb.3c00302 Risa Sasaki 1 , Shogo Toda 1 , Takaiku Sakamoto 2 , Eiji Sakuradani 2 , Shinsuke Shigeto 1
Affiliation
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Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution–alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of ∼7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear–nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids.
中文翻译：
使用线性和非线性拉曼显微光谱法同时成像和表征单个 Aurantiochytrium limacinum 细胞中的多不饱和脂肪酸、类胡萝卜素和微晶鸟嘌呤
破囊壶菌是异养海洋原生生物，以其对健康有益的各种化合物（例如多不饱和脂肪酸和类胡萝卜素）的高生产能力而闻名。尽管很多努力都集中在开发高效微生物生产的最佳培养方法上，但这些高价值化合物及其相互关系在单细胞水平上并没有得到很好的理解。在这里，我们使用自发（线性）拉曼和多重相干反斯托克斯拉曼散射 (CARS) 显微光谱法来可视化和表征单活 Aurantiochytrium limacinum 中积累的脂质（例如，二十二碳六烯酸）和类胡萝卜素（例如，虾青素）细胞。借助多元曲线分辨率的自发拉曼成像 - 交替最小二乘法使我们能够对我们检测到的分子成分进行明确的分配，并分别推导出它们的细胞内分布。近红外激发 CARS 成像产生的拉曼图像至少比自发拉曼成像快一个数量级，同时抑制了类胡萝卜素的贡献。随着培养时间从 2 天增加到 5 天，脂质量增加了约 7 倍，而类胡萝卜素量没有显着变化。此外，我们在A. limacinum中观察到一个高度局部化的成分细胞。该成分被发现是鸟嘌呤和其他嘌呤衍生物的混合晶体。本研究展示了线性-非线性拉曼混合方法的潜力，该方法允许以无标记方式进行准确的分子鉴定和快速成像，以将来自单细胞的信息与有用破囊壶菌的大规模培养策略联系起来。
更新日期：2023-03-15
中文翻译：
使用线性和非线性拉曼显微光谱法同时成像和表征单个 Aurantiochytrium limacinum 细胞中的多不饱和脂肪酸、类胡萝卜素和微晶鸟嘌呤
破囊壶菌是异养海洋原生生物，以其对健康有益的各种化合物（例如多不饱和脂肪酸和类胡萝卜素）的高生产能力而闻名。尽管很多努力都集中在开发高效微生物生产的最佳培养方法上，但这些高价值化合物及其相互关系在单细胞水平上并没有得到很好的理解。在这里，我们使用自发（线性）拉曼和多重相干反斯托克斯拉曼散射 (CARS) 显微光谱法来可视化和表征单活 Aurantiochytrium limacinum 中积累的脂质（例如，二十二碳六烯酸）和类胡萝卜素（例如，虾青素）细胞。借助多元曲线分辨率的自发拉曼成像 - 交替最小二乘法使我们能够对我们检测到的分子成分进行明确的分配，并分别推导出它们的细胞内分布。近红外激发 CARS 成像产生的拉曼图像至少比自发拉曼成像快一个数量级，同时抑制了类胡萝卜素的贡献。随着培养时间从 2 天增加到 5 天，脂质量增加了约 7 倍，而类胡萝卜素量没有显着变化。此外，我们在A. limacinum中观察到一个高度局部化的成分细胞。该成分被发现是鸟嘌呤和其他嘌呤衍生物的混合晶体。本研究展示了线性-非线性拉曼混合方法的潜力，该方法允许以无标记方式进行准确的分子鉴定和快速成像，以将来自单细胞的信息与有用破囊壶菌的大规模培养策略联系起来。
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