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

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 β 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. 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 β 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. ERRATAJournal of Lipid ResearchVol. 51Issue 4PreviewThe authors of “Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans” (J. Lipid Res. 51: 672–677) have advised the Journal that the figure legend for Figure 2 of their manuscript is incorrect. It should read as follows: Full-Text PDF Open Access With short lifespan, rapid reproduction cycle, amenable genetics, and a remarkable conservation of human disease genes and pathways, Caenorhabditis elegans has become an ideal model to study the mechanisms of disease pathogenesis (1Kaletta T. Hengartner M.O. Finding function in novel targets: C. elegans as a model organism.Nat. Rev. Drug Discov. 2006; 5: 387-398Crossref PubMed Scopus (756) Google Scholar). Being optically transparent, C. elegans has been extensively employed to visualize lipid storage with fluorescent imaging of lipids stained with Nile Red (2Ashrafi K. Chang F.Y. Watts J.L. Fraser A.G. Kamath R.S. Ahringer J. Ruvkun G. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes.Nature. 2003; 421: 268-272Crossref PubMed Scopus (838) Google Scholar, 3Wang M.C. O’Rourke E.J. Ruvkun G. Fat metabolism links germline stem cells and longevity in C. elegans.Science. 2008; 322: 957-960Crossref PubMed Scopus (289) Google Scholar) or recently with label-free single-frequency coherent anti-Stokes Raman scattering (CARS) imaging (4Hellerer T. Axang C. Brackmann C. Hillertz P. Pilon M. Enejder A. Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy.Proc. Natl. Acad. Sci. USA. 2007; 104: 14658-14663Crossref PubMed Scopus (278) Google Scholar). However, either fluorescent or single-frequency CARS imaging lacks spectral information critical for lipid composition analysis. Although spontaneous Raman microscopy (5van Manen H.J. Kraan Y.M. Roos D. Otto C. Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes.Proc. Natl. Acad. Sci. USA. 2005; 102: 10159-10164Crossref PubMed Scopus (273) Google Scholar) and multiplex CARS microscopy (6Rinia H.A. Burger K.N.J. Bonn M. Muller M. Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy.Biophys. J. 2008; 95: 4908-4914Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) could analyze the composition of single lipid droplets, their image acquisition speeds are too slow for live-cell or animal study. Alternatively, third harmonic generation microscopy can visualize lipid droplets using optical heterogeneity of biological samples for contrast mechanism (7Debarre D. Supatto W. Pena A.M. Fabre A. Tordjmann T. Combettes L. Schanne-Klein M.C. Beaurepaire E. Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy.Nat. Methods. 2006; 3: 47-53Crossref PubMed Scopus (474) Google Scholar). However, third harmonic generation lacks chemical selectivity to analyze lipid composition. Consequently, a microscopy tool to study critical aspects of lipid metabolism, including lipid storage and composition, is lacking. By employing a multifunctional microscope that permits high-speed CARS imaging, two-photon excited fluorescence (TPEF) imaging, and confocal Raman spectral analysis with a picosecond laser source (8Wang H.W. Le T.T. Cheng J.X. Label-free imaging of arterial cells and extracellular matrix using a multimodal CA