Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent antiStokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-␤) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.lipid metabolism ͉ nonlinear microscopy ͉ obesity

Adiponectin is an adipokine with insulin-sensitising actions in vertebrates. Its receptors, AdipoR1 and AdipoR2, are PAQR-type proteins with 7-transmembrane domains and topologies reversed that of GPCR's, i.e. their C-termini are extracellular. We identified three adiponectin receptor homologs in the nematode C. elegans, named paqr-1, paqr-2 and paqr-3. These are differently expressed in the intestine (the main fat-storing tissue), hypodermis, muscles, neurons and secretory tissues, from which they could exert systemic effects. Analysis of mutants revealed that paqr-1 and -2 are novel metabolic regulators in C. elegans and that they act redundantly but independently from paqr-3. paqr-2 is the most important of the three paqr genes: mutants grow poorly, fail to adapt to growth at low temperature, and have a very high fat content with an abnormal enrichment in long (C20) poly-unsaturated fatty acids when combined with the paqr-1 mutation. paqr-2 mutants are also synthetic lethal with mutations in nhr-49, sbp-1 and fat-6, which are C. elegans homologs of nuclear hormone receptors, SREBP and FAT-6 (a Δ9 desaturase), respectively. Like paqr-2, paqr-1 is also synthetic lethal with sbp-1. Mutations in aak-2, the C. elegans homolog of AMPK, or nhr-80, another nuclear hormone receptor gene, suppress the growth phenotype of paqr-2 mutants, probably because they restore the balance between energy expenditure and storage. We conclude that paqr-1 and paqr-2 are receptors that regulate fatty acid metabolism and cold adaptation in C. elegans, that their main function is to promote energy utilization rather than storage, and that PAQR class proteins have regulated metabolism in metazoans for at least 700 million years.

Zebrafish are an increasingly popular vertebrate model organism in which to study biological phenomena. It has been widely used, especially in developmental biology and neurobiology, and many aspects of its development and physiology are similar to those of mammals. The popularity of zebrafish relies on its relatively low cost, rapid development and ease of genetic manipulation. Moreover, the optical transparency of the developing fish together with novel imaging techniques enable the direct visualization of complex phenomena at the level of the entire organism. This potential is now also being increasingly appreciated by the lipid research community. In the present review we summarize basic information on the lipid composition and distribution in zebrafish tissues, including lipoprotein metabolism, intestinal lipid absorption, the yolk lipids and their mobilization, as well as lipids in the nervous system. We also discuss studies in which zebrafish have been employed for the visualization of whole-body lipid distribution and trafficking. Finally, recent advances in using zebrafish as a model for lipid-related diseases, including atherosclerosis, obesity, diabetes and hepatic steatosis are highlighted. As the insights into zebrafish lipid metabolism increase, it is likely that zebrafish as a model organism will become an increasingly powerful tool in lipid research.

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We introduce dual-pump coherent anti-Stokes-Raman scattering (dual-CARS) microscopy. This new technique permits simultaneous imaging of two species characterized by different molecular vibrations, as well as the removal of nonresonant background. This is achieved by using three synchronized laser pulses probing two different vibrations. We demonstrate the virtues of the method by imaging a mixture of nondeuterated and deuterated lipids, clearly distinguishing the individual components and their organization in the mixed arrangement. Further, dual-CARS images of lipid stores in living Caenorhabditis elegans nematodes show that the suppression of the nonresonant background results in significantly enhanced image contrast.

The effects of different preparation methods on the bioaccessibility of β-carotene in orange-fleshed sweet potato (OFSP), an important food crop in sub-Saharan Africa, have been evaluated using an in vitro digestion procedure. The preparation methods included, on fresh roots, boiling followed by puréeing and oil addition (BOL) and homogenization followed by boiling and oil addition (HOM); on milled flour from freeze-dried fresh roots, cooking of porridge followed by oil addition (POA) and oil addition to flour followed by cooking of porridge (POB). The retention of all-trans-β-carotene ranged from 58% (POB) to 72% (BOL). The presence of oil during heating resulted in a significantly higher formation of 13-cis-β-carotene for the POB-treated samples than for the other samples. The efficiency of micellarization of all-trans-β-carotene after in vitro digestion was 50% (HOM), 48% (POB), 31% (POA), and 16% (BOL). Brightfield microscopy of the cell structure after processing and in vitro digestion showed a high degree of cell-wall rupture for the HOM-treated samples, whereas cells appeared intact for the BOL samples. Also, coherent anti-Stokes Raman scattering (CARS) microscopy showed smaller β-carotene bodies residing in the HOM samples than in the BOL samples after digestion. These results suggest that the in vitro bioaccessibility of β-carotene in an OFSP meal can be improved by processing methods that promote cell-wall rupture.

Cellulose perforated by micro-channels (Ø ~500 μm) has been investigated as a potential future scaffold material for meniscus implants. Scaffolds seeded with 3T6 fibroblasts were cultivated with mechanical stimulation in a compression bioreactor for enhanced collagen production. Constructs under dynamic compression at a frequency of 0.1 Hz and compression strain of 5% were compared to static cultures used as controls. The three-dimensional distributions of collagen fibers and fibroblasts in the cellulose scaffolds were studied under native, soft-matter conditions by combined second harmonic generation and coherent antiStokes Raman scattering microscopy, requiring no artificial sample preparation. Results showed that the micro-channels facilitated the alignment of cells and collagen fibers and that collagen production was enhanced by mechanical stimulation. Thus, cell-seeded, micro-channeled cellulose scaffolds provided guided tissue growth required to obtain an ultrastructure mimicking that of the meniscus.

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