Dataset Information

Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans.

ABSTRACT: The ubiquity of lipids in biological structures and functions suggests that lipid metabolisms are highly regulated. However, current invasive techniques for lipid studies prevent characterization of the dynamic interactions between various lipid metabolism pathways. Here, we describe a noninvasive approach to study lipid metabolisms using a multifunctional coherent anti-Stokes Raman scattering (CARS) microscope. Using living Caenorhabditis elegans as a model organism, we report label-free visualization of coexisting neutral and autofluorescent lipid species. We find that the relative expression level of neutral and autofluorescent lipid species can be used to assay the genotype-phenotype relationship of mutant C. elegans with deletions in the genes encoding lipid synthesis transcription factors, LDL receptors, transforming growth factor beta receptors, lipid desaturation enzymes, and antioxidant enzymes. Furthermore, by coupling CARS with fingerprint confocal Raman analysis, we analyze the unsaturation level of lipids in wild-type and mutant C. elegans. Our study shows that complex genotype-phenotype relationships between lipid storage, peroxidation, and desaturation can be rapidly and quantitatively analyzed in a single living C. elegans.

SUBMITTER: Le TT

PROVIDER: S-EPMC2817597 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans.

Le Thuc T TT Duren Holli M HM Slipchenko Mikhail N MN Hu Chang-Deng CD Cheng Ji-Xin JX

Journal of lipid research 20090923 3

The ubiquity of lipids in biological structures and functions suggests that lipid metabolisms are highly regulated. However, current invasive techniques for lipid studies prevent characterization of the dynamic interactions between various lipid metabolism pathways. Here, we describe a noninvasive approach to study lipid metabolisms using a multifunctional coherent anti-Stokes Raman scattering (CARS) microscope. Using living Caenorhabditis elegans as a model organism, we report label-free visual ...[more]

PMID: 19776402

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Project description: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.

Dataset Information

Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans.

Publications

Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans.

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