A Comparative Study of Fat Storage Quantitation in Nematode Caenorhabditis elegans Using Label and Label-Free Methods
A Comparative Study of Fat Storage Quantitation in Nematode Caenorhabditis elegans Using Label and Label-Free Methods
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DOI：
10.1371/journal.pone.0012810
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发表时间：
2010-09-16
期刊：
影响因子：
3.7
通讯作者：
Tissenbaum, Heidi A.
中科院分区：
文献类型：
--
作者：
Yen, Kelvin;Le, Thuc T.;Tissenbaum, Heidi A.
由于线虫能量代谢途径的保守性，线虫已被用作研究人类肥胖的模式生物。测定C.在线虫中，已经使用了许多脂溶性染料，包括BODIPY、尼罗红、油红O和苏丹黑。然而，染料标记的检测结果往往与C.优雅的。另一种分析C.最近已经用相干反斯托克斯拉曼散射（汽车）显微术描述了线虫。在这里，我们比较了汽车显微镜与标准染料标记技术和生化定量分析野生型C脂肪储存的性能。elegans，并且在胰岛素/IGF-1信号传导途径中具有基因突变，包括基因daf-2（胰岛素/IGF-1受体）、rict-1（rictor）和sgk-1（血清糖皮质激素激酶）。汽车成像提供了一种直接测量脂肪储存的前所未有的细节，包括总脂肪储存以及单个脂滴的大小，数量和脂链不饱和度。此外，汽车/TPEF成像揭示了存在于皮下组织和肠细胞中的中性脂质物质以及仅存在于肠细胞中的自发荧光细胞器。重要的是，来自C-H丰富的中性脂质的汽车场的相干添加允许脂肪储存的选择性汽车成像，并且自发拉曼分析的进一步耦合提供了前所未有的细节，包括单个脂滴的脂链不饱和度。我们观察到，尽管daf-2、rict-1和sgk-1突变体会影响胰岛素/IGF-1信号传导，但它们在中性脂质和自发荧光物质方面表现出截然不同的表型。我们发现，汽车成像给出类似于标准生化甘油三酯定量的定量。此外，我们独立地证实，喂养蠕虫与活体染料不会导致染色的脂肪储存，而是隔离染料在溶酶体相关的细胞器。相比之下，固定剂染色方法提供了可重复的数据，但由于自体荧光物质的干扰和除脂肪储存外的细胞结构的非特异性染色而易于出错。重要的是，在比较C. elegans脂质储存综上所述，我们证实汽车显微镜提供了一种直接，非侵入性和无标记的手段来定量分析活C。优美的
The nematode Caenorhabditis elegans has been employed as a model organism to study human obesity due to the conservation of the pathways that regulate energy metabolism. To assay for fat storage in C. elegans, a number of fat-soluble dyes have been employed including BODIPY, Nile Red, Oil Red O, and Sudan Black. However, dye-labeled assays produce results that often do not correlate with fat stores in C. elegans. An alternative label-free approach to analyze fat storage in C. elegans has recently been described with coherent anti-Stokes Raman scattering (CARS) microscopy. Here, we compare the performance of CARS microscopy with standard dye-labeled techniques and biochemical quantification to analyze fat storage in wild type C. elegans and with genetic mutations in the insulin/IGF-1 signaling pathway including the genes daf-2 (insulin/IGF-1 receptor), rict-1 (rictor) and sgk-1 (serum glucocorticoid kinase). CARS imaging provides a direct measure of fat storage with unprecedented details including total fat stores as well as the size, number, and lipid-chain unsaturation of individual lipid droplets. In addition, CARS/TPEF imaging reveals a neutral lipid species that resides in both the hypodermis and the intestinal cells and an autofluorescent organelle that resides exclusively in the intestinal cells. Importantly, coherent addition of the CARS fields from the C-H abundant neutral lipid permits selective CARS imaging of the fat store, and further coupling of spontaneous Raman analysis provides unprecedented details including lipid-chain unsaturation of individual lipid droplets. We observe that although daf-2, rict-1, and sgk-1 mutants affect insulin/IGF-1 signaling, they exhibit vastly different phenotypes in terms of neutral lipid and autofluorescent species. We find that CARS imaging gives quantification similar to standard biochemical triglyceride quantification. Further, we independently confirm that feeding worms with vital dyes does not lead to the staining of fat stores, but rather the sequestration of dyes in lysosome-related organelles. In contrast, fixative staining methods provide reproducible data but are prone to errors due to the interference of autofluorescent species and the non-specific staining of cellular structures other than fat stores. Importantly, both growth conditions and developmental stage should be considered when comparing methods of C. elegans lipid storage. Taken together, we confirm that CARS microscopy provides a direct, non-invasive, and label-free means to quantitatively analyze fat storage in living C. elegans.