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 anti-Stokes 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-beta) 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.

Fig. 1.

Comparison of CARS and fluorescence microscopy. (A) Autoscaled CARS and (B) two-photon fluorescence images (80 × 80 μm², 20-s integration time) of a Nile red-stained daf-4 mutant (C. elegans) arrested in its dauer stage for 3 weeks. The pharynx can be distinguished in the upper left corner. While the CARS image shows the full distribution of lipid droplets, also the hypodermal collection, the two-photon fluorescence image (excitation at 1,064 nm) merely shows the lipid droplets in the intestine. Thus, the hypodermal droplets cannot be visualized by means of fluorescence microscopy. This limitation is also clear from the evaluation of the lipid volume fractions for the corresponding z-stacks, which resulted in 23% for the CARS image and a misleading number of 16% for the fluorescence image.

Fig. 2.

CARS spectra of C. elegans. Series of CARS images were collected of N2 L4, N2 L1, pha-3, daf-2, and dauer daf-4 nematodes in vivo simultaneously with a reference image of glass (no resonant features) for normalization while tuning the molecular vibration probed. The normalized mean CARS intensity of the lipid droplets, scaled between 0 and 1 and shifted along the y axis, is plotted vs. the vibration, forming CARS spectra shown in A. A tissue matrix spectrum accompanies the dauer spectrum, indicating sampling frequency and standard deviations. All lipid droplet spectra exhibit a peak at 2,845 cm⁻¹, corresponding to the symmetric CH₂ vibration, forming optimal image contrast. In addition, wild-type (L1 and L4) and pha-3 nematodes show a resonance at the asymmetric CH₂ vibration (2,880 cm⁻¹), which cannot be distinguished in the daf-2 (data not shown) or -4 dauer spectra. This indicates that a shift in lipid ordering occurs from gel to liquid phase when extensive lipid storage is promoted. Two representative images of an N2 L4 larva are shown: resonant (2,845 cm⁻¹) (B) and nonresonant (2,790 cm⁻¹) (C).

Fig. 3.

Quantitative analysis of CARS microscopy images of C. elegans. Representative images (42 × 42 μm²) of different mutants at the L4 larval stage are shown (intensity profiles are shown in SI Fig. 6). Compared with the wild-type (A), the pha-3 in B generates notably weaker CARS signals, whereas the daf-2 (L4, 25°C) in C as well as the daf-4 dauer in D exhibits significantly higher CARS intensities and lipid droplet density. The images were all collected during 20 s, with the excitation powers 7.5 (1,064 nm) + 15 (817 nm) mW. The number of CARS photons registered is color-coded according to the color bar shown. A schematic drawing of a C. elegans larva is shown in E, illustrating the geometry of data collection and image analysis with the image planes of the CARS z-stack covering the central part of the larva. The average volume fraction of lipid stores in the indicated ellipsoidal volume of interest is shown in the diagram in F for the different categories together with the fraction found in the hypodermal cells. Compared with the wild type, significantly lower lipid volume fractions are obtained for the pha-3 mutant and higher for the daf-2 and -4 dauer categories. The energy storage of C. elegans arrested in its dauer stage not only is limited to the intestinal cells but also takes place to a large extent in the hypodermal cells.

Fig. 4.

CARS microscopy volume images. Normalized CARS volume images (B, D, F, and H) were reconstructed from z-stacks, represented by one of the optical sections shown in A, C, E, and G (A, 58 × 58 μm²; C, 26 × 26 μm²; E, 45 × 45 μm²; and G, 50 × 50 μm²). In A and B, a clear alignment of the lipid droplets is visualized in a hatching larva along its ventral cord, as indicated by the arrow, most likely remaining yolk granules. This regular structure can be recognized in the L1 larva (N2) in C and D, complemented by a droplet collection distributed in the entire intestinal cells. The dark areas lacking lipid droplets (arrows) are most likely the intestinal nuclei. With larval development, the distribution of lipid droplets becomes more dominant in the intestinal cells, as shown for the L4 larva (N2) in E and F. For the daf-4 dauer mutant in G and H, the small-sized droplets in the hypodermal cells can clearly be distinguished from the large-sized droplets in the intestinal cells.