672 Journal of Lipid Research Volume 51, 2010
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
With short lifespan, rapid reproduction cycle, amena-
ble genetics, and a remarkable conservation of human
disease genes and pathways, Caenorhabditis elegans has be-
come an ideal model to study the mechanisms of disease
pathogenesis ( 1) . Being optically transparent, C. elegans
has been extensively employed to visualize lipid storage
with fl uorescent imaging of lipids stained with Nile Red
( 2, 3) or recently with label-free single-frequency coher-
ent anti-Stokes Raman scattering (CARS) imaging ( 4) .
However, either fl uorescent or single-frequency CARS im-
aging lacks spectral information critical for lipid composi-
tion analysis. Although spontaneous Raman microscopy
( 5) and multiplex CARS microscopy ( 6) could analyze the
composition of single lipid droplets, their image acquisi-
tion speeds are too slow for live-cell or animal study. Alter-
natively, third harmonic generation microscopy can
visualize lipid droplets using optical heterogeneity of bio-
logical samples for contrast mechanism ( 7) . However,
third harmonic generation lacks chemical selectivity to
analyze lipid composition. Consequently, a microscopy
tool to study critical aspects of lipid metabolism, includ-
ing lipid storage and composition, is lacking. By employ-
ing a multifunctional microscope that permits high-speed
CARS imaging, two-photon excited fl uorescence (TPEF)
imaging, and confocal Raman spectral analysis with a pi-
cosecond laser source ( 8, 9) , we demonstrate the capabil-
ity to study the dynamic interactions between lipid storage,
peroxidation, and desaturation in wild-type and mutant
C. elegans .
Abstract The ubiquity of lipids in biological structures
and functions suggests that lipid metabolisms are highly
regulated. However, current invasive techniques for lipid
studies prevent characterization of the dynamic interac-
tions between various lipid metabolism pathways. Here, we
describe a noninvasive approach to study lipid metabolisms
using a multifunctional coherent anti-Stokes Raman scatter-
ing (CARS) microscope. Using living Caenorhabditis elegans
as a model organism, we report label-free visualization of
coexisting neutral and autofl uorescent lipid species. We
fi nd that the relative expression level of neutral and auto-
fl uorescent lipid species can be used to assay the genotype-
phenotype relationship of mutant C. elegans with deletions
in the genes encoding lipid synthesis transcription factors,
LDL receptors, transforming growth factor ? receptors,
lipid desaturation enzymes, and antioxidant enzymes . Fur-
thermore, by coupling CARS with fi ngerprint confocal
Raman analysis, we analyze the unsaturation level of lipids
in wild-type and mutant C. elegans . Our study shows that
complex genotype-phenotype relationships between lipid
storage, peroxidation, and desaturation can be rapidly and
quantitatively analyzed in a single living C. elegans . —Le,
T. T., H. M. Duren, M. N. Slipchenko, C-D. Hu, and J-X.
Cheng. Label-free quantitative analysis of lipid metabolism
in living Caenor habditis elegans. J. Lipid Res . 2010. 51:
Supplementary key words cholesterol synthesis • coherent anti-Stokes
Raman scattering • lipid desaturation • label-free imaging • lipid stor-
age • peroxidation • spontaneous Raman spectroscopy • two-photon
excited fl uorescence
This work is supported by a National Institutes of Health postdoctoral fellowship
(F32HL089074 to T.T.L.), National Institutes of Health Grant R01EB007243
(to J-X.C.), and National Science Foundation Grant MCB0420634 (to
C-D.H.). Its contents are solely the responsibility of the authors and do not
necessarily represent the offi cial views of the National Institutes of Health.
T.T. L. and H.M.D. designed and performed experiments. T.T.L. and M.N.S.
analyzed data. J-X.C. and C-D.H. contributed analytical tools, reagents, and
research guidance. T.T.L and H.M.D wrote the article. All authors read and
approved the fi nal manuscript.
Manuscript received 31 July 2009 and in revised form 29 September 2009.
Published, JLR Papers in Press, September 29, 2009
Label-free quantitative analysis of lipid metabolism in
living Caenorhabditis elegans
Thuc T. Le, * Holli M. Duren, † Mikhail N. Slipchenko, * Chang-Deng Hu, 1,†,§ and
Ji-Xin Cheng 1, * ,§, **
Weldon School of Biomedical Engineering,* Department of Medicinal Chemistry and Molecular
Pharmacology, † Purdue Cancer Center, § and Department of Chemistry,** Purdue University , West Lafayette,
Abbreviations: CARS, coherent anti-Stokes Raman scattering;
RME-2, receptor-mediated endocytosis 2; SREBP-1, sterol regulatory
element binding protein 1; TPEF, two-photon excited fl uorescence.
1 To whom correspondence should be addressed.
e-mail: firstname.lastname@example.org (C-D.H.); jcheng@purdue.
The online version of this article (available at http://www.jlr.org )
contains supplementary data in the form of three fi gures and one
at PURDUE UNIV LIB TSS on February 19, 2010
Supplemental Material can be found at:
Label-free imaging of lipid metabolism673
5(tm420)/fat-7(wa36) ], CE833 [ sbp-1(ep176) ], FX776 [ sod-
1(tm776) ], CB1364 [ daf-4(e1364) ], and DH1390 [ rme-2(b1008) ].
Except for CE833 [ sbp-1(ep176) ], which was cultured at 15°C, all
C. elegans strains were cultured on agar plates seeded with OP50
Escherichia coli at 20°C.
Experimental details of gas chromatography and mass spec-
trometry analysis of fatty acids composition have been previously
described ( 10) . Cholesterol, phosphatidylcholine, and superox-
ide dismutase were performed using commercially available kits
according to manufacturer’s protocols (catalog numbers
10007640, 10009926, and 706002; Cayman Chemical, Ann Arbor,
MI). To collect worms, 16 cm plates were washed with M9 media
into Eppendorf tubes. Worms were further washed multiple
times with M9 media to remove any residual bacteria. The ex-
pression level of phosphatidylcholine was used as a reference to
adjust for the number of C. elegans analyzed.
By simultaneous CARS and TPEF imaging of living C.
elegans , we discovered two distinctive lipid species ( Fig. 1 ;
see supplementary Video I). One lipid species can be visu-
alized by CARS only, which we identify as neutral lipid
( Fig. 1A ). Visualization of such neutral lipid species with
CARS has been previously reported by Hellerer et al. ( 4) .
The other lipid species can be visualized by both autofl uo-
rescent signals identifi able with TPEF imaging and lipid
signals identifi able with CARS imaging ( Fig. 1B, C ). Fur-
ther confocal Raman spectral analyses of the neutral lipid
droplets reveal strong chemical signatures typical of tria-
A multifunctional CARS microscope
A spectrometer with a 300 g/mm grating and a thermoelectri-
cally cooled back-illuminated EMCCD (Newton 920-BRD; Andor
Technology, Belfast, Ireland) is mounted to the side port of a la-
ser scanning microscope (IX71/FV300; Olympus, Center Valley,
PA) to allow TPEF imaging, CARS imaging, and spontaneous
Raman spectral analysis on the same platform. Pump and Stokes
lasers are tuned to 14,140 cm ? 1 and 11,300 cm ? 1 , respectively, to
be in resonance with the CH 2 symmetric stretch vibration at 2,840
cm ? 1 . The combined beams are focused into the sample through
a ×60 water immersion microscope objective with a 1.2 numerical
aperture. Forward-detected CARS signal is collected by an air
condenser with a 0.55 numerical aperture, transmitted through a
600/65 nm band-pass fi lter, and detected by a photomultiplier
tube (H7422-40; Hamamatsu, Japan). Simultaneously, back-
refl ected TPEF signal is collected by the same illuminating objec-
tive, spectrally separated from the excitation source, transmitted
through a 520/40 nm band-pass fi lter, and detected by a photo-
multiplier tube (H7422-40; Hamamatsu) mounted at the back
port of the microscope. Following CARS and TPEF imaging, the
Stokes beam is blocked and the pump laser-induced Raman scat-
tering signal is directed toward the spectrometer to permit spec-
tral analysis from 830 to 3,100 cm ? 1 , which covers both the
fi ngerprint and the CH-stretch vibration regions. Due to the uti-
lization of a confocal pinhole with a diameter of 50 µm before
the spectrometer, the Raman signals arise from a focal volume of
2.3 femtoliter at the center of the fi eld of view of a CARS image.
Average acquisition time for a 512 × 512 pixel CARS image is 1.12
s, and a full-spectral Raman analysis is 4 s. The combined Stokes
and pump laser power is kept constantly at 40 mW. For all Raman
spectral measurements, pump laser power is reduced to 10 mW.
Variability in Raman spectral measurements of neutral lipid
droplets is discussed in supplementary Figs. II and III . Experi-
mental details of Rama spectra acquisition and data analysis were
previously described by Slipchenko et al. ( 9).
Imaging conditions and data analysis
All C. elegans were anesthetized in a droplet of 100 mM sodium
azide and mounted on fresh 2% agarose slides prior to imaging.
To evaluate the expression level of neutral and autofl uorescent
lipid droplets, we defi ne a probed volume with xyz dimensions of
125 × 125 × 35 µm. We fi rst locate the midsection of an adult wild-
type or mutant C. elegans and then perform simultaneous depth
imaging with CARS and TPEF along the vertical (z) axis at 1 µm
step size to obtain 36 frames. Background CARS and TPEF pixel
intensity are subtracted from average pixel intensity of CARS and
TPEF signals of the probed volume to obtain the expression level
of neutral and autofl uorescent lipid droplets, respectively. Back-
ground CARS and TPEF pixel intensity are defi ned as the average
pixel intensity of probed volumes devoid of neutral and autofl uo-
rescent lipid droplets. The background CARS signal includes sig-
nals arising from the worm bodies. The expression levels of neutral
and autofl uorescent lipid droplets are adjusted to 1 for the wild
type and comparatively for mutant C. elegans . Because CARS signal
is quadratically dependent on the concentration of CH 2 molecular
vibration at ? p ? ? S = 2,840 cm ? 1 , we present the square root value
of CARS signal as the expression level of neutral lipid droplets.
C. elegans strains
All C. elegans strains were obtained from the Caenorhabditis
Genetics Center: wild-type Bristol (N2), BX107 [ fat-5(tm420) ],
BX106 [ fat-6(tm331) ], BX153 [ fat-7(wa36) ], BX160 [ fat-5(tm420)/
fat-6(tm331) ], BX156 [ fat-6(tm331)/fat-7(wa36) ], BX110 [ fat-
Fig. 1. Chemical imaging and spectral analysis of lipid species in
C. elegans . CARS (A; red) and TPEF (B; blue) imaging of a larval L2
C. elegans . Images presented as a three-dimensional projection of
25 frames taken along vertical axis at 1 µm intervals. C: An enlarged
and overlaid image of lipid species in C. elegans . D: Spontaneous
Raman spectral analysis of neutral lipid droplets (red) and auto-
fl uorescent lipid droplets (blue).
at PURDUE UNIV LIB TSS on February 19, 2010
674Journal of Lipid Research Volume 51, 2010
has also been reported by Hellerer et al. ( 4) . However, the
effect of daf-4 deletion on the decrease of autofl uorescent
lipid droplet level is reported here for the fi rst time. Fi-
nally, the fi fth phenotype is observed with rme-2 mutants,
where a 1.3-fold increase in the expression of neutral lipid
droplets is accompanied by a wild-type level expression of
autofl uorescent lipid droplets ( Fig. 2A–C ). This pheno-
type suggests a possible role of RME-2 in neutral lipid
droplet formation. However, this relationship has not
been elucidated in the current literature. Taken together,
our results show that the relative expression levels of neu-
tral and autofl uorescent lipid species provide a reliable
means to assay for phenotypes of C. elegans mutants.
Using biochemical assays, we fi nd that changes in the
level of autofl uorescent lipid species in fat-5/fat-6 , sbp-1 ,
and sod-1 mutants are due to distinct mechanisms ( Fig.
2D ). By measuring the cholesterol level in total lipid ex-
tracts, we fi nd a 1.2-fold increase in fat-5/fat-6 mutants, a
4-fold reduction in sbp-1 mutants, and no change in sod-1
mutants. These observations suggest that ? 9 desaturases
and SREBP-1 are involved in cholesterol biosynthesis,
whereas superoxide dismutase is not. Therefore, increases
in autofl uorescent lipid species observed in fat-5/fat-6 and
sbp-1 mutants are likely due to a different mechanism com-
pared to sod-1 mutants ( Fig. 2D ).
By CARS imaging and spontaneous Raman analysis of
single lipid droplets, we further evaluated the degree of
lipid-chain unsaturation in wide-type and mutant C. ele-
gans . Using C18 fatty acid methyl esters as standards, we
show that the degree of lipid-chain unsaturation can be
measured using three Raman-active bands, including 1280
cm ? 1 , 1660 cm ? 1 , and 3015 cm ? 1 ( Fig. 3A–C ) ( 6, 21) . Be-
cause the signal-to-noise ratio for C=C stretch is highest at
1660 cm ? 1 band, we select this band to evaluate lipid chain
unsaturation ( 6) . We observe that I 1660 /I 1445 is linearly cor-
related with lipid chain unsaturation ( Fig. 3C ). Using
I 1660 /I 1445 as a reliable measure of ? 9 desaturase enzymatic
activity, we systematically evaluated lipid chain unsatura-
tion of neutral lipid droplets. We observed signifi cant re-
duction in C=C stretch vibration signal in ? 9 desaturase
mutants compared to wild-type C. elegans ( Fig. 3D ). Quan-
titative analysis of lipid droplet I 1660 /I 1445 in six desaturase
mutants reveals up to 2-fold reduction in lipid chain un-
saturation in single and double ? 9 desaturase mutants
( Fig. 3E ). Our Raman spectral analyses are further sup-
ported by GC-MS measurements of lipid chain unsatura-
tion of total lipid extracts (see supplementary Fig. I ). We
fi nd a dramatic decrease in the ratios of unsaturated oleic,
linoleic, and eicosenoic fatty acids over saturated stearic
acid in fat-5/fat-6 and fat-6/fat-7 mutants compared to the
wild type ( Fig. 3F ). Complete analyses of lipid composition
of ? 9 desaturase mutants using GC-MS have been de-
scribed previously ( 10) . However, unlike GC-MS, the com-
bination of CARS imaging and confocal Raman spectral
analysis, so called compound Raman microscopy ( 6) , en-
abled us to measure lipid chain desaturation noninvasively
with single lipid droplet sensitivity. This capability should
allow real-time dynamic studies of the activity of desaturases
and other lipid metabolism enzymes in living C. elegans .
cylglycerides ( Fig. 1D ). In contrast, the fl uorescence from
the autofl uorescent lipid droplets dominates the Raman
spectra ( Fig. 1D ). Several previous studies have also identi-
fi ed such autofl uorescent particles and associated them with
lipids, oxidative stress, and lifespan of C. elegans ( 11, 12) .
To explore the potential of using the neutral and auto-
fl uorescent lipid species as a readout of lipid metabolism,
we evaluated their expression levels in wild-type and mu-
tant C. elegans . All selected C. elegans mutants have been
well characterized, with deletions in the genes encoding
lipid metabolism proteins, including ? 9 desaturases
(palmitoyl-CoA desaturase fat-5 and stearoyl-CoA desatu-
rases fat-6 and fat-7 ) ( 10) , sterol regulatory element bind-
ing protein ( sbp-1 ) ( 13, 14) , copper/zinc superoxide
dismutase ( sod-1 ) ( 15, 16) , type II transforming growth
factor ? receptor ( daf-4 ) ( 4, 17) , and LDL receptor ( rme-
2 ) 18 . Specifi cally, ? 9 desaturases are lipogenic enzymes
critical for the conversion of saturated fatty acids into
monounsaturated fatty acids ( 19, 20) . Sterol regulatory el-
ement binding protein 1 (SREBP-1) is a transcription fac-
tor that controls the expression of lipogenic enzymes ( 14) .
Superoxide dismutase is an antioxidant enzyme that pro-
tects cells from reactive oxygen species ( 15) . Type II trans-
forming growth factor ? receptor is a transmembrane
serine/threonine kinase whose functions are implicated
in many biological processes, including the insulin signal-
ing pathway ( 17) . Receptor-mediated endocytosis 2 (RME-
2) is an LDL receptor that mediates yolk endocytosis and
fatty acid transport in oocytes ( 18) . Although the func-
tions of these lipid metabolism proteins are well under-
stood, their roles in the expression level of autofl uorescent
lipid species in C. elegans have not been characterized.
Our analyses of neutral and autofl uorescent lipid drop-
let expression level in C. elegans mutants reveal fi ve distinc-
tive phenotypes as compared to the wild type ( Fig. 2A ).
The fi rst phenotype is observed in C. elegans mutants with
double deletion of ? 9 desaturases, fat-5/fat-6 , where a 1.4-
fold decrease in the expression level of neutral lipid drop-
lets is accompanied by a 3-fold increase in the expression
level of autofl uorescent lipid droplets ( Fig. 2A–C ). This
phenotype is consistent with previous biochemical analy-
ses where a decrease in fat storage and an increase in the
expression of genes involved in fatty acid oxidation are ob-
served in ? 9 desaturase mutants ( 10 ). The second pheno-
type is observed in sbp-1 mutants, where there is a near
complete suppression of both neutral and autofl uorescent
lipid droplet expression ( Fig. 2A–C ). This phenotype is
also supported by the established roles of SREBP-1 in cho-
lesterol and fatty acids homeostasis ( 14) . The third pheno-
type is observed with sod-1 mutants, where the wild-type
level of neutral lipid droplet expression is accompanied by
a 2-fold increase in autofl uorescent lipid droplet expres-
sion ( Fig. 2A–C ). This phenotype suggests a direct role of
antioxidant enzymes in regulating the level of autofl uores-
cent lipid droplets. The fourth phenotype is observed with
daf-4 mutants, where a 1.4-fold increase in neutral lipid
droplet is accompanied by a 2-fold reduction in autofl uo-
rescent lipid droplet compared to the wild type ( Fig. 2A–
C ). This increase in neutral lipid storage in daf-4 mutants
at PURDUE UNIV LIB TSS on February 19, 2010
Label-free imaging of lipid metabolism 675
cent lipid droplet formation. These observations suggest
that SREBP-1, ? 9 desaturases, and transforming growth
factor ? receptor participate in shared pathways by both
neutral and autofl uorescent lipid droplet formation. In
contrast, deletion of antioxidant enzymes affects only
autofl uorescent lipid droplet formation, and deletion of
LDL receptor RME-2 affects only neutral lipid droplet
formation. These observations suggest that antioxidant
enzymes or LDL receptor RME-2 participates in specifi c
autofl uorescent or neutral lipid droplet formation path-
ways, respectively. Thus, the relationship between the ex-
pression levels of neutral and autofl uorescent lipid species
could potentially be used to identify the involvement of
unknown proteins in lipid metabolism pathways.
In addition to visualization of neutral and autofl uorescent
lipid species with CARS and TPEF signals, spontaneous
In this study, we report label-free visualization and quan-
titation of coexisting neutral and autofl uorescent lipid
species in living C. elegans . We show that multimodal imag-
ing allows rapid genotype-phenotype screening of lipid
metabolism in C. elegans . Specifi cally, we fi nd that the
expression of neutral and autofl uorescent lipid species
are dynamically correlated to specifi c genes. Deletion of
SREBP-1 transcription factor for lipid and cholesterol syn-
thesis suppresses both neutral and autofl uorescent lipid
droplet formation. Deletion of ? 9 desaturases represses
neutral lipid-droplet formation and promotes autofl uores-
cent lipid droplet formation. Conversely, deletion of trans-
forming growth factor ? receptor represses autofl uorescent
lipid droplet formation and promotes neutral autofl uores-
Fig. 2. Expression levels of lipid species in the wild type and mutant C. elegans . A: CARS imaging of lipid
(red) and TPEF imaging of autofl uorescent lipid species (blue) of adult N2 wild-type and mutant C. elegans.
Images are presented as three-dimensional projections of 36 frames taken along the vertical axis at 1 µm
intervals. Expression levels of neutral lipid droplets (B) and autofl uorescent lipid droplets (C) as a function
of wild-type and mutant worms. Expression levels are normalized to 1 for the wild type and comparatively for
mutants. Error bars represent distribution six adult wild-type or mutant C. elegans . D: Biochemical measure-
ments of cholesterol level and superoxide dismutase (SOD) activity. Expression levels are normalized to 1 for
the wild type and comparatively for mutant C. elegans . Error bars represent distribution across three repeated
at PURDUE UNIV LIB TSS on February 19, 2010
676 Journal of Lipid Research Volume 51, 2010
Lipids play a ubiquitous role in human physiology.
Membrane lipids actively regulate cell proliferation, apop-
tosis, migration, and senescence ( 25) . Lipid-mediated en-
docrine networks regulate systemic metabolic homeostasis
( 26) . Excessive lipid storage in obesity is associated with
increased risk factors for diabetes, cardiovascular diseases,
stroke, and cancer ( 27) . Given the signifi cance of lipids in
biology, lipid metabolism should be thoroughly and sys-
tematically studied. The multifunctional CARS microscope
described in this article, when combined with recent ad-
vances in genetics ( 28) and high-throughput screening
( 29) for C. elegans research, should enable functional stud-
ies of lipid metabolism enzymes, interaction of lipid me-
tabolism networks, and discovery of new lipid metabolism
pathways. Because CARS microscopy has been applied for
in vivo imaging ( 30–32) , our lipid metabolism studies in
living C. elegans should be extensible to both animals and
humans. The versatility of the multifunctional CARS mi-
croscope would render it an indispensible tool to the study
of lipids in diseases.
The authors thank Han-Wei Wang for help with experiments.
Raman microspectroscopy enables noninvasive quantita-
tion of desaturation in single lipid droplets. In general,
lipid storage, peroxidation, and desaturation are all criti-
cal to the health of animals. Indeed, lipid peroxidation is
strongly linked to the lifespan of animals ( 22) . Loss of
stearoyl-CoA desaturase-1 function has been shown to re-
duce body adiposity, increase insulin sensitivity, and resis-
tance to diet-induced adiposity in mice ( 19) . However, loss
of stearoyl-CoA desaturase-1 function is also associated
with increased aorta atherosclerosis ( 23) and infl amma-
tion ( 24) . Nonetheless, the impacts of lipid desaturation
on lipid storage, peroxidation, or infl ammation are not
clearly understood. Herein, we report that genetic dele-
tions of ? 9 desaturase genes in C. elegans are strongly as-
sociated with a reduction of lipid chain unsaturation ( Fig.
3E ) and neutral lipid storage ( Fig. 2B ) as well as a signifi -
cant increase in autofl uorescent lipid species ( Fig. 2C )
and cholesterol synthesis ( Fig. 2D ). Given the strong con-
servation in lipid metabolism from C. elegans to humans
( 1) , it is conceivable that future in-depth investigation of
lipid desaturation in C. elegans could bring new insights to
the roles of desaturases in human health and diseases.
Fig. 3. Analysis of lipid chain unsaturation in ? 9 desaturase wild-type and mutant C. elegans . Raman spectra of fatty acid methyl esters: stea-
rate (C18:0), oleate (C18:1), and linoleate (C18:2) at fi ngerprint region from 1,200 to 1,800 cm ? 1 (A) and the CH-stretch vibration region
from 2,800 to 3,100 cm ? 1 (B). C: I 1660 /I 1445 as a function of lipid chain unsaturation of stearate, stearate:oleate 1-to-1 mixture, oleate,
oleate:linoleate 1-to-1 mixture, and linoleate. Error bars represent distribution across three repeated experiments. D: Representative Raman
spectra of neutral lipid droplets in wild-type and mutant C. elegans . E: Quantitative analyses of lipid chain unsaturation of lipid droplets in wild-
type and ? 9 desaturase mutants. Error bars represent distribution across 18 lipid droplets measured in six adult wild-type or mutant C. elegans .
F: GC-MS analysis of fatty acids composition of wild-type and ? 9 desaturase double mutants. The ratio of unsaturated fatty acids over C18:0 is
normalized to 1 for the wild type and comparatively for mutants. Error bars represent distribution across three repeated experiments.
at PURDUE UNIV LIB TSS on February 19, 2010
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at PURDUE UNIV LIB TSS on February 19, 2010