Researchers at Chalmers University of Technology and at Göteborg University, both in Göteborg, Sweden, have developed a method for using coherent anti-Stokes Raman scattering (CARS) microscopy to quantify the storage of fat in Caenorhabditiselegans, a nematode frequently studied as a model for systems in higher-order organisms. Of the roughly 400 genes associated with lipid storage in C. elegans, most have human counterparts. A better understanding of how the nematode stores fat could lead to useful knowledge about human fat storage.Researchers used a coherent anti-Stokes Raman scattering microscope to probe the C-H vibration at 2845 cm–1 and create an optical section (A) of lipid stores in a C. elegans daf-4 dauer mutant. By stacking optical sections, they can reconstruct a 3-D image (B) of lipids stored as large-size droplets in the intestinal cells but also as small-size droplets in the hypodermal cells. Image reprinted with permission of PNAS.The researchers — led by Annika Enejder and Marc Pilon — studied wild-type and pha-3, daf-2 and daf-4 mutant C. elegans. The pha-3 has a feeding defect that allows studies under starved conditions. The daf-2 partially activates the dauer formation, a survival strategy in which the worms store fat, slow their metabolism and stop maturing. The daf-4 allows researchers to control when the worm activates the dauer formation.The scientists compared coherent anti-Stokes Raman scattering with conventional fluorescence microscopy to visualize lipids in the worms, concluding that the former created a more accurate image.Coherent anti-Stokes Raman scattering microscopy can probe for specific groups of chemicals, can be used for three-dimensional sectioning and does not require a fluorescent reporter molecule. However, the technique does require a well-optimized setup, and because of its technically advanced level, it requires specialist competence, Enejder explained. The setup included a coherent anti-Stokes Raman scattering microscope built on a Nikon inverted microscope. For excitation, the researchers used an Nd:Vanadate laser operating at 1064 nm with a 7-ps pulse duration and a 76-MHz repetition rate to pump two intracavity-doubled APE Levante optical parametric oscillators, both from High Q Laser Production GmbH of Hohenems, Austria. For image acquisition, they used a Hamamatsu photomultiplier tube combined with a Becker & Hickl single-photon counting device.The researchers used the Raman technique to image lipid stores in the different worms at various larval stages and found that the daf-2 mutant has total lipid stores that are about 1.4 times that of the wild type, and that the daf-4 stores nearly double that of the wild type. After three weeks of arrested development as dauer larvae, the daf-4 mutants still had 1.6 times more lipid stores than the wild type. The pha-3 never achieves lipid stores comparable to those of the wild type, which leads to stunted growth. In addition, the researchers discovered that besides having more lipid stores, the daf-2 and daf-4 mutants also tend to store more lipids in a liquid form. “This really begs the question of whether this is an important part of the accumulation of extra lipid stores,” Enejder said. “Could a liquid order be more suitable to long-term storage?” Enejder and Pilon now plan to investigate how worms grown under different conditions store fat and whether this mechanism has relevance for the human metabolism. “Some of the questions that we would like to address include: Do worms that experience a period of starvation early in life adjust their life strategy by storing more fat so as to prepare for other periods of starvation? Do mutations that affect fat storage fall into specific classes?” For example, are there mutations that affect only the number or size of the droplets, or mutations that affect only the hypodermal stores?Lastly, Enejder said that the researchers hope to discover mechanisms of fat storage regulation that have human counterparts, an investigation that could help address the obesity epidemic that is plaguing the US and some parts of Western Europe.PNAS, Sept. 11, 2007, pp. 14658–14663.