As C. elegans
is increasingly used as a model system to study the basic mechanisms of fat storage and energy homeostasis, it is critical to ensure that the established assays accurately determine and reflect fat content. To that effect, here we study the performance of existing techniques including vital and fixative labeling and label-free assays to evaluate fat stores. We find that vital staining with the fat-soluble dyes Nile Red and BODIPY labeled fatty acids cannot serve as proxies for fat stores. Nile Red and BODIPY-labeled fatty acids do not stain fat stores but stain autofluorescent organelles previously identified as LROs in living worms 
. It is possible that Nile red and BODIPY-labeled fatty acids are actively transported into intestinal cells. In fact, a specific transporter for Nile Red has been identified in yeast 
. Similar transporters in worms may sequester Nile Red and BODIPY-labeled fatty acids, thus shielding them from the fat stores in living worms. Furthermore, Clokey and Jacobson reported over two decades ago that exogenous fluorescent probes are taken up by endocytosis and accumulate within the autofluorescent lipofuscin granules, which are the secondary lysosomes and active recipients of endocytosed fluorescent probes 
. Our findings, together with other independent reports, strongly suggest that vital staining using fat-soluble dyes including both Nile Red and BODIPY, should not be used to assay fat stores in living C. elegans
We find that fixative staining using Sudan Black, Oil Red O, and Nile Red produce consistent and reproducible data on fat stores (). Qualitatively, fixative staining data agree with CARS imaging data on the level of fat stores in mutant daf-2
relative to wild type worms. However, our fixative staining data differ with CARS imaging data and biochemical analysis on the level of fat stores in rict-1
mutants where there is a high level of autofluorescent gut granules/lipofuscin. These results are in contrast to those found by Soukas et al. 
and are further discussed in Text S1
. Since CARS microscopy provides a direct measure of fat stores instead of a proxy of fat storage, it is possible that this difference could be attributable to errors introduced during fixation or staining procedures. Additional error could also be due to the following reasons. First, lipofuscin is a hydrophobic accumulation of highly oxidized cellular proteins and lipids that have characteristic fluorescent properties 
. Their hydrophobic properties pose a problem for lipophilic stains because both Sudan Black and Oil Red O stain for lipofuscin as well as triglycerides 
. As Nile Red is also a hydrophilic stain, it is possible that it could also stain lipofuscin. Therefore, fixative staining cannot distinguish neutral fat stores from lipofuscin. Second, the autofluorescence of lipofuscin might contribute to errors in the quantitation of fluorescent stains such as Nile Red and BODIPY. Third, formaldehyde fixation of the worms introduces additional non-specific fluorescence due to the fluorescent properties of the Schiff bases produced 
. Finally, non-specific staining of dyes, variable dye concentration, and thoroughness of washing procedures are among the other factors that might contribute to the inaccuracy in assaying for fat stores using labeling techniques 
. Therefore, although fixative staining offers a simple and reproducible means to assay fat stores in C. elegans
, we suggest that errors may arise due to interference from autofluorescence granules and non-specific staining of lipofuscin and other cellular organelles. Thus, fixative staining to assay for fat stores in C. elegans
should also present data on autofluorescent granules.
Qualitative comparison of fat stores in mutants relative to wild type worms.
In contrast to labeling techniques, label-free CARS imaging provides a direct and reliable means to assay fat stores in living C. elegans
. In addition, CARS imaging allows for detailed analysis of the number, size, and lipid-chain unsaturation of single lipid droplets. Multimodal CARS and TPEF imaging further allows simultaneous visualization of neutral lipids and autofluorescent lipofuscins. Together, CARS microscopy allows for the assaying of various aspects of fat storage in living C. elegans
in a non-invasive and label-free manner. Fat storage in the form of lipid droplets has recently emerged as a complex process that is dependent on insulin signaling, phospholipid synthesis, fatty acids synthesis and desaturation, fatty acids uptake and transport, activation of nuclear hormone receptors, and upregulation of a wide range of lipid synthesis enzymes 
. Genetic mutations of genes that control lipid metabolism in C. elegans
and other organisms have been shown to impact the total fat stores, as well as the size, number, and composition of lipid droplets 
. Furthermore, a strong correlation between fat storage, oxidative stress, and the lifespan of C. elegans
has been revealed in recent years 
. Therefore, the versatility of CARS microscopy is critical not only for forward genotype-phenotype screening of genes that control fat accumulation, but also for the studies of potential interactions of lipid metabolism genes. Most importantly, CARS microscopy is currently the only reliable method to assay for fat stores in living C. elegans
. When combined with recent advances in microfluidic devices 
, this capability should allow dynamic studies of the correlation between lipid metabolism, behavioral response, and aging over the lifetime of a single worm.
We have used several methods including fixative stains, live stains and live imaging to analyze C. elegans
fat storage. Our data supports previous publications that vital dyes should be used with caution for lipid analysis in C. elegans
. Using CARS microscopy we show Nile Red itself stains the autofluorescent granules and this is the confounding problem. Importantly, we show that similar stages and growth conditions should be used when comparing different methods (Figure S5
). We suggest that CARS is the optimal method for C. elegans
fat storage. However, when not available, we suggest using one of the fixative methods as well as checking autofluorescent levels.