Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy

Abstract

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

Proteomic Study and Marker Protein Identification of Caenorhabditis elegans Lipid Droplets

“…As a direct LD detection, our approach may also be used to assess whether the Raman signals of live C. elegans captured by Coherent anti-Stokes Raman Spectroscopy and Stimulated Raman Spectroscopy come from LDs or other types of organelles (37)(38)(39) Raman Spectroscopy may determine whether these signals can reliably act as a proxy for TAG levels in live C. elegans. Furthermore, an in vitro assay using isolated LDs and other cellular organelles may help to resolve the issue of the specificity of the Raman signals that are detected by Coherent anti-Stokes Raman Spectroscopy and Stimulated Raman Spectroscopy.…”

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

“…CARS microscopy images fatty acid molecules directly by exciting specifically the C-H bonds in which they are rich and is therefore a dye-independent method to quantitatively visualize lipid droplets with great spatial resolution (21). CARS microscopy could detect no difference between fluvastatin-treated worms or control worms; the fraction of the sampled volume occupied by lipid droplets did not vary between controls and statin-treated worms ( Fig.…”

“…CARS Microscopy. The CARS microscopy analysis was performed as previously described (21), with the modification that the area sampled corresponded to the first intestinal ring, just posterior to the pharynx, and the use of a slightly different setup (see SI Materials and Methods).…”

Vibrational spectroscopy as a tool for studying drug-cell interaction: Could high throughput vibrational spectroscopic screening improve drug development?

“…[93,94] Many studies have been carried out on model organisms such as Caenorhabditis elegans [94,95], showing the ability of these techniques to image lipids with high contrast. Hellerer et al [95] report on the study of lipid storage in Caenorhabditis elegans using CARS and how genetic variations affect this. There is potential for these techniques to be extended to study how lipid storage and metabolism is altered in response to drug treatment.…”