Figure 1.
Simplified diagram of insulin/IGF signaling.
Bolded proteins are proteins that have been investigated in this study.
Figure 2.
Label and label-free imaging methods to assay fat storage in C. elegans.
(A) Labeled imaging of fat stores using Sudan Black, Oil Red O, and Nile Red staining of fixed worms and Nile Red and BODIPY-labeled fatty acids fed to live worms. (B) Label-free visualization of neutral lipid species and autofluorescent gut granules using simultaneous CARS and TPEF imaging, respectively. Note the co-localization of TPEF signal with CARS signal in the intestinal cells. Images are presented as 3-D stacks of 30 frames taken at 1 µm increment along the vertical axis. Rightmost panel is an enlargement of the overlaid image with the xy dimensions of 20 µm×20 µm. It should be noted that the association of fluorescent signal with lipid signal can be found surrounding, at one end, above, or below the lipid signal. In addition, the lipid contents of the fluorescent particles vary from one to another. For the particular image presented, the fluorescent puncta do exhibit lipid signal when examined at a higher magnification.
Figure 3.
Quantitative analysis of the total fat stores and the size, number, and composition of lipid droplets with CARS microscopy.
(A) Total fat stores are defined as the integrated CARS signal intensity over the probed volume minus the background signal arising from worm bodies (leftmost panel). The ImageJ software is used for binary image conversion of CARS images and for particle analysis to determine the size and number of lipid droplets. Upper limits (2 µm2) can be manually set to minimize sizing errors due to lipid droplets being too close to each other. (B) The compositions of six lipid droplets (crosshairs) are analyzed using spontaneous Raman spectroscopy. Statistically insignificant variability in lipid composition is observed for lipid droplets within a single worm. Leftmost panel is an enlargement of Figure 2A with the xy dimensions of 20 µm×20 µm.
Figure 4.
Label and label-free imaging of fat storage in wild type and mutant C. elegans.
(A) Visualization of fat stores in fixed worms using fixative dyes Sudan Black, Oil Red O, and Nile Red*. Arrows indicate the pharynx (B) Visualization of fat stores (upper panels) and autofluorescent gut granules (lower panels) using simultaneous CARS and TPEF imaging of living worms, respectively. Images are presented as 3-D stacks of 30 frames taken at 1 µm increment along the vertical axis. *Nile Red pictures were taken with a more sensitive black and white camera.
Figure 5.
Quantitative analysis of fat storage in wild type and mutant C. elegans.
(A) Total fat stores in mutant worms relative to wild type worms assayed by feeding worms with vital dyes Nile Red and BODIPY-labeled fatty acids, with CARS imaging, and with fixative staining with Nile Red. Data represent the average of 3 independent trials with an average of 18 worms quantified per trial for fed Nile Red, 20 worms quantified per trial for fed BODIPY, 9 worms quantified per trial for CARS imaging, and 23 worms quantified per trial for Nile Red on fixed worms. Data are normalized to 100 for wild type worms and comparatively for mutant worms. Error bars represent the standard error. (B) Quantitative analysis of autofluorescent granules using TPEF imaging and size, number, and lipid unsaturation of lipid droplets using CARS imaging. TPEF data are normalized to 100 for wild type worms and comparatively for mutant worms. Lipid unsaturation represents the ratio of C = C peak intensity over C-C peak intensity, or I1660/I1445. Data represent the average of 3 independent trials with lipid droplets of 9 worms quantified per trial, or 27 worms total. Error bars represent the standard error.
Figure 6.
Possible errors in dye-labeled assays to analyze fat stores in living C. elegans.
(A) Emission spectra of autofluorescent granules, BODIPY, and Nile Red obtained with microspectrometry. The autofluorescent granules and BODIPY are excited at 457 nm using an Argon-Ion continuous-wave laser. The Nile Red is excited with a 543 nm Helium-Neon continuous-wave laser. (B) Insignificant bleed-through of autofluorescent granule signal is observed using a 600 nm bandpass filter and two-photon excitation at 885 nm. (C) CARS and TPEF imaging of two rict-1 worms fed with Nile Red. Autofluorescent granules (blue) are detected with a 520 nm bandpass filter and Nile Red (red) is detected with a 600 nm bandpass filter. Pink color is the result of overlapping blue and red colors which indicates co-localization of autofluorescent granules and Nile Red. Note the complete co-localization of autofluorescent granules and Nile Red in worm 1 (upper panels) and partial co-localization of autofluorescent granules and Nile Red in worm 2 (lower panels). Rightmost panels are enlargements of the CARS/Nile Red overlaid images with the xy dimensions of 20 µm×20 µm. Note the poor co-localization of lipid droplets (grey) and Nile Red (red). Images are presented as 3-D stacks of 30 frames taken at 1 µm increment along the vertical axis.
Table 1.
Qualitative comparison of fat stores in mutants relative to wild type worms.