Department of Electronic and Computer Engineering , Hong Kong University of Science and Technology , Clear Water Bay , Kowloon , Hong Kong 999077 , China.
Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study , Hong Kong University of Science and Technology , Clear Water Bay , Kowloon , Hong Kong 999077 , China.
Anal Chem. 2019 Feb 5;91(3):2279-2287. doi: 10.1021/acs.analchem.8b04875. Epub 2019 Jan 10.
Quantitative methods to precisely measure cellular states in vivo have become increasingly important and desirable in modern biology. Recently, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool to visualize small biological molecules tagged with alkyne (C≡C) or carbon-deuterium (C-D) bonds in the cell-silent region. In this study, we developed a technique based on SRS microscopy of vibrational tags for quantitative imaging of lipid synthesis and lipolysis in live animals. The technique aims to overcome the major limitations of conventional fluorescent staining and lipid extraction methods that do not provide the capability of in vivo quantitative analysis. Specifically, we used three bioorthogonal lipid molecules (the alkyne-tagged fatty acid 17-ODYA, deuterium-labeled saturated fatty acid PA-D, and unsaturated fatty acid OA-D) to investigate the metabolic dynamics of lipid droplets (LDs) in live Caenorhabditis elegans ( C. elegans). Using a hyperspectral SRS (hsSRS) microscope and subtraction method, the interfering non-Raman background was eliminated to improve the accuracy of lipid quantification. A linear relationship between SRS signals and fatty acid molar concentrations was accurately established. With this quantitative analysis tool, we imaged and determined the changes in concentration of the three fatty acids in LDs of fed or starved adult C. elegans. Using the hsSRS imaging mode, we also observed the desaturation of fatty acids in adult C. elegans via spectral analysis on the SRS signals from LDs. The results demonstrated the unique capability of hsSRS microscopy in quantitative analysis of lipid metabolism in vivo.
定量方法在现代生物学中，精确测量细胞状态变得越来越重要和必要。最近，受激拉曼散射（SRS）显微镜已成为一种强大的工具，可以可视化标记炔烃（C≡C）或碳氘（C-D）键的小分子生物在细胞静默区。在这项研究中，我们开发了一种基于 SRS 显微镜振动标记的技术，用于对活体动物中脂质合成和脂肪分解进行定量成像。该技术旨在克服传统荧光染色和脂质提取方法的主要局限性，这些方法不具备活体定量分析的能力。具体而言，我们使用三种生物正交脂质分子（炔烃标记的脂肪酸 17-ODYA、氘标记的饱和脂肪酸 PA-D 和不饱和脂肪酸 OA-D）来研究活体秀丽隐杆线虫（C. elegans）中脂滴（LDs）的代谢动力学。通过使用高光谱 SRS（hsSRS）显微镜和减法方法，可以消除干扰的非拉曼背景，从而提高脂质定量的准确性。准确建立了 SRS 信号与脂肪酸摩尔浓度之间的线性关系。有了这个定量分析工具，我们可以对喂食或饥饿的成年 C. elegans 中 LDs 中的三种脂肪酸的浓度变化进行成像和测定。使用 hsSRS 成像模式，我们还通过对 LDs 中 SRS 信号进行光谱分析，观察到成年 C. elegans 中脂肪酸的去饱和作用。结果表明，hsSRS 显微镜在活体脂质代谢定量分析中具有独特的能力。
