Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy.

Despite the ubiquitous roles of lipids in biology, the detection of lipids has relied on invasive techniques, population measurements, or nonspecific labeling. Such difficulties can be circumvented by a label-free imaging technique known as coherent anti-Stokes Raman (CARS) microscopy, which is capable of chemically selective, highly sensitive, and high-speed imaging of lipid-rich structures with submicron three-dimensional spatial resolution. We review the broad applications of CARS microscopy to studies of lipid biology in cell cultures, tissue biopsies, and model organisms. Recent technical advances, limitations of the technique, and perspectives are discussed.

Lipids play a critical role in human health and diseases. Phospholipids, glycolipids, and sterol lipids are the major components of the cell membrane ( 1 ). Sphingolipids constitute up to 80% of the myelin sheaths, which are the membranous structures that wrap around the axons for insulation and for proper propagation of electrical impulses ( 2 ). Glycerolipids or triglycerides serve as the cytoplasmic energy depots ( 3 ). Bioactive lipids including sphingolipid second messengers, second messengers from phosphotidylinositol, lipid activators of G-protein coupled receptors, and lipid activators of nuclear receptors regulate a wide range of biological processes including cell death, cell proliferation, senescence, migration, and infl ammation ( 4,5 ). Dysregulation of lipid metabolism in hyperlipidemia or obesity can lead to many debilitating diseases including diabetes, atherosclerosis, and neurodegenerative diseases ( 6,7 ). biological systems has been widely demonstrated (23)(24)(25). Most importantly, with the beating frequency ( p Ϫ s ) tuned to c.a. 2845 cm Ϫ 1 that matches the symmetric stretch vibration of CH 2 groups, a strong CARS signal is produced from lipid-rich structures that are abundant in CH 2 groups. With such capability, CARS microscopy has found broad applications in the fi eld of lipid biology in recent years ( 26,27 ). In this review, we describe the basic principle of CARS microscopy and its recent development to biologists who are unfamiliar with CARS technology. Successful applications of CARS microscopy to the studies of lipids in health and diseases are summarized and discussed. In particular, we highlight the innovative studies of two important lipid-rich structures, cytoplasmic lipid droplets and axonal myelin sheath.

HISTORY OF DEVELOPMENT
The phenomenon of coherent Raman scattering was fi rst reported by scientists at the Ford Motor Co. in 1965 ( 28 ). Since the introduction in the early 1970s ( 29 ), CARS spectroscopy has been widely used as a spectroscopic tool ( 30 ). The fi rst application of CARS to imaging was demonstrated in 1982 with a noncollinear beam geometry by scientists at the Naval Research Laboratory ( 31 ). Nearly two decades later, CARS microscopy was revisited with a collinear beam confi guration at the Pacifi c Northwest National Laboratory in 1999 ( 32 ). Since then, the CARS renaissance has stimulated further development and applications by researchers from broad scientifi c disciplines. Continuous contributions from many research groups over the last decade have vastly improved the design, stability, and sensitivity of CARS microscopy [for reviews, see ( 18,(23)(24)(25)].

ESSENTIAL FEATURES OF A HIGH-SPEED CARS MICROSCOPE
In this section we describe a high-speed, multifunctional CARS microscope ( Fig. 1 ) that is ready for biological applications. This platform results from theoretical study ( 22 ) and multi-step technical advances ( 18,25 ) of CARS microscopy. Important features of the microscope are described in the sections below.

Near IR laser excitation
The near IR lasers avoid two-photon electronic enhancement of the nonresonant background ( 32 ), reduce the photodamage induced by multiphoton absorption ( 33 ), and diminish tissue scattering leading to increased optical penetration depth ( 34 ). As a two-beam modality, CARS microscopy is mostly operated with picosecond (ps) pulses, either from two synchronized Ti:sapphire lasers ( 35 ) or from a synchronously pumped Optical Parametric Oscillator system ( 34 ). Picosecond pulse excitation not only provides suffi cient spectral resolution ( 36 ) but also increases the ratio of resonant signal to nonresonant background ( 35 ). Although tunable ps laser shifts associated with the molecular vibration and give rise to a Raman spectrum specifi c to the molecule. Because each molecule has its unique Raman signature, Raman scattering has been widely employed to study the intrinsic chemical composition of biological structures without the need for labeling (15)(16)(17). However, the applications of Raman scattering to biological imaging are faced with many technical challenges. Following light-matter interaction, approximately one in ten million photons undergoes Raman scattering. The spontaneous Raman cross-section is ‫ف‬ 10 Ϫ 30 cm 2 per molecule as compared with the fl uorescence cross-section of ‫ف‬ 10 Ϫ 16 cm 2 per molecule ( 15 ). Weak Raman signal requires long integration time of ‫ف‬ 100 millisecond per pixel for image acquisition, or hours for an image of 512 by 512 pixels. This timescale is unsuitable for imaging dynamic living biological systems. In addition, autofl uorescence intrinsic to many biological structures at visible wavelength is stronger than Raman scattering. In such cases, informative Raman spectral details are masked by the fl uorescence.
Fortunately, the Raman scattering signal can be enhanced by several orders of magnitude with a nonlinear optical (NLO) method called coherent anti-Stokes Raman scattering (CARS) microscopy ( 18 ). CARS is a fourwave mixing process in which three laser fi elds at the pump ( p ), Stokes ( s ), and probe ( ′ p ) frequencies interact with a medium to generate a new fi eld at the anti-Stokes frequency as = ( p Ϫ s ) + ′ p . In most experiments, the pump fi eld and probe fi eld come from the same laser beam. CARS signal arises from any medium with nonzero third-order susceptibility ( 19 ). The signal is enhanced when the beating frequency ( p Ϫ s ) is in resonance with a molecular vibration frequency, which provides the vibrational contrast in a CARS image. Owing to the coherent addition of the CARS radiations, the CARS intensity is quadratically dependent on the number of the vibrational oscillators in the focal volume. In the samples with high concentration of vibrational oscillators, CARS signal is many folds stronger than spontaneous Raman scattering signal, which is linearly dependent on the number of vibrational oscillators. The large signal level in CARS microscopy enables high-speed imaging, which is important for live cell and tissue studies. By scanning the lasers of high repetition rate (in MHz), an image acquisition speed at one frame per second has been achieved ( 20 ) and has been recently increased to twenty frames per second ( 21 ). The high imaging speed not only avoids respiration-induced image distortion during in vivo imaging, but also allows real-time inspection of biological processes. Moreover, the NLO excitation ensures that the CARS signal is only generated at the center of the focus, offering CARS microscopy an inherent 3-D spatial resolution. The lateral and axial resolutions with a 60× water immersion objective were measured to be 0.23 and 0.75 m ( 22 ), which allows detection of subcellular structures in a tissue environment.
With the developments of CARS imaging platforms, high-speed three-dimensional chemical imaging of living lipid bodies or myelin sheath in cultured cells or sliced tissues ( 40,41 ). For objects with an axial length much smaller than the excitation wavelengths, the phase matching condition is fulfi lled in both forward and backward directions, which constitute the fi rst mechanism for epi-detected CARS (E-CARS) ( 42 ). E-CARS also arises from the discontinuity of (3) at an interface ( 22 ) and back-scattering of forward CARS photons ( 21 ). E-CARS is important for live animal imaging where the epi-detected signal arises from backscattering of the forward propagating CARS photons by the tissue ( 21,43 ).

Laser-scanning on a confocal microscope platform
The most signifi cant advantage of CARS over spontaneous Raman is its large signal level to allow for high-speed vibrational imaging. This advantage has been realized by scanning pulsed lasers of high repetition rates (in MHz), which resulted in an image acquisition speed of a frame per second ( 20 ). Video rate imaging can be realized by scanning the laser with a polygon mirror or a resonant scanner ( 21,44 ).

Multimodal NLO imaging
An important advantage of the CARS microscope is that other NLO modalities, including TPEF and SFG, can be implemented on the same platform. Multimodality is important because different NLO imaging methods have their distinctive advantages: TPEF can be used to visualize proteins and ions with fluorescent labeling or specific autofluorescent structures; SFG is selective to noncentrosymmetric molecular assemblies, such as collagen fibrils and crystals; and CARS is naturally sensitive to lipid-rich structures such as adipocytes. The pulsed laser beams for CARS could be simultaneously used for TPEF and electronic SFG imaging. For instance, Fu et al. ( 45 ) demonstrated multimodal NLO imaging of ex vivo spinal tissues by CARS imaging of myelin sheath, SFG imaging of astrocyte processes, and TPEF imaging of calcium indicators on the same platform. Technically, one can separate the CARS and TPEF signals based on their spectral and coherence properties. The CARS photons are highly directional in the forward and can be collected with an air condenser of low numerical aperture (NA). The fluorescence is spectrally broad and incoherent; it is therefore negligible after the narrow bandpass CARS filters and the air condenser in the forward channel. Due to the Gouy phase shift, the forward SFG signal is deflected from the optical axis ( 46 ) and is fairly weak if a low-NA air condenser is used. Instead, the backward SFG and TPEF photons can be effectively collected by the high NA laser-focusing objective. Examples of CARS-based multimodal NLO imaging of lipid-rich structures are shown in Fig. 2 .

Coupling CARS imaging with spontaneous Raman spectroscopy
It has been realized that the CARS and spontaneous Raman have their distinctive advantages; CARS permits systems operating in the near IR range are widely accepted for high-speed CARS imaging [for reviews, see ( 18,23,25 )], the reduced effi ciency of NLO process caused by longer pulse duration ( 37 ) hinders wide application of ps lasers to two-photon excited fl uorescence (TPEF) and second harmonic generation (SHG) imaging. Therefore, toward the goal of coupling the CARS imaging modality to other widely used platforms of multiphoton microscopy, Pegoraro et al. ( 38 ) and Chen et al. ( 39 ) showed independently high-speed CARS imaging of lipids with femtosecond (fs) laser pulses. Commercial CARS microscopes with either ps or fs laser sources are now available through Leica and Olympus, respectively.

Collinear beam geometry
Non-collinear beam geometry was used in CARS spectroscopy to fulfi ll the phase matching condition ( 30 ). When the interaction length became very small ( ‫ف‬ 1 m) under the tight focusing condition in microscopy, the phase matching condition was fulfi lled with a collinear beam geometry for forward-detected CARS (F-CARS) ( 32 ). The collinear beam geometry greatly simplifi ed the optical alignment and has been a key step in producing high-quality CARS images.

Forward-detection versus epi-(i.e., backward-) detection
For objects that are comparable or larger than the wavelengths of incident lasers, the CARS signal goes forward as a result of constructive addition of radiations ( 18 ). Importantly, because the CARS signal is highly directional in the forward direction, an air condenser is suffi cient for collection of the F-CARS signal. F-CARS is suitable for imaging PMT1 and PMT2 are detectors for F-CARS and E-CARS, respectively. The pump laser is also used for generating spontaneous Raman signals from an object visualized by CARS. A spectrometer with a pinhole attached to the side port of the microscope is used for confocal recording of spontaneous Raman spectra. Although not shown, sum frequency generation (SFG) and two-photon excited fl uorescence (TPEF) imaging can be performed on the same platform. PH, pinhole; DM, dichroic mirror; L, lense; PMT, photomultiplier tube.

Study of lipid droplet biology
Lipid droplets (LDs) have been well observed as a prominent cytoplasmic structure ( 49 ). However, the studies of LDs have been hindered by a long held perception of LDs as inert energy depots. Contrary to this perception, increasing evidence points to an active role of LDs in biological processes ( 50 ). In addition to storing triglycerides high-speed imaging by focusing the energy on a single Raman band whereas spontaneous Raman permits complete fi ngerprint analysis at a pixel of interest. Recently, a compound Raman microscope has been demonstrated by using the same ps laser source for high-speed coherent Raman imaging of a sample and confocal Raman spectral analysis of lipid-rich structures such as sebaceous gland ( 47 ) and fat in Caenorhabditis elegans ( 48 ). Examples of CARS imaging and Raman spectral analysis of lipid-rich structures are shown in Fig. 3 . C: CARS image of two lipid-rich foam cells. D: CARS image of lipid-rich intestinal cells of a C57BL/6J mouse after feeding. E: Overlaid CARS and SFG image of myelin sheaths (red) and astroglial processes (blue). Image is a courtesy of Terry B. Huff. F: Overlaid CARS and TPEF image of neutral lipid droplets (red) and autofl uorescent lipid species (blue) in a living C. elegans . Scale bars: 5 µm. Images adapted from references ( 48,59,90,98 ). been serving as a model organism to study the pathogenesis of metabolic diseases ( 61 ). In C. elegans , lipids are stored in the form of LDs mostly in the intestinal cells and to some extent in the hypodermal cells ( 63 ). A wide range of lipid synthesis enzymes, including desaturases and elongases, are present in C. elegans , which account for a full range of saturated, monounsaturated, and polyunsaturated fatty acids of various lengths ( 63 ). Genetic deletions of the lipid synthesis enzymes generally lead to quantifiable phenotypes, including changes in the quantity and composition of lipids, stunted growth, altered movement, reduced body size, defective neurotransmission, and shortened lifespan ( 64,65 ). Therefore, C. elegans is a desirable model for the integrative studies of how a specifi c lipid synthesis enzyme impacts the physiology, metabolism, and behavior of an organism.
However, the studies of lipid metabolism in C. elegans are hindered by several technical challenges. First, the labeling effi ciency of LDs in C. elegans can vary from worm to worm. Labeling effi ciency is also lower in live worms than in fi xed worms ( 66 ). In addition, LDs of the intestinal cells are labeled but not LDs of the hypodermal cells ( 67 ). Thus, visualization and quantitation of LDs in C. elegans are subject to large technical errors. Second, the fl uorescent spectra of labeling dyes overlap with the fl uorescent spectrum of autofl uorescent particles. The expression of autofl uorescent particles in C. elegans is strongly correlated to oxidative stress and aging ( 68,69 ). Thus, the studies of LDs with labeling mask information, which could be critical to the evaluation of C. elegans physiology. Third, the analysis of LD composition is currently performed using gas chromatography ( 70 ). This measurement approach provides a population average value and masks the individual variability intrinsic to worms. Furthermore, the dynamic correlation between lipid composition and C. elegans behavior is not possible with bulk measurement techniques.
CARS microscopy presents an unprecedented opportunity to study lipid metabolism in living C. elegans . In the fi rst application of CARS microscopy to C. elegans , Hellerer et al. ( 67 ) monitored changes in the lipid packing state of LDs as a function of larvae developmental stages. Hellerer et al. also examined lipid storage in wild-type and mutants of C. elegans with genetic deletions of proteins involved in the feeding behavior and insulin signaling pathway. In another study, Morck et al. ( 71 ) used CARS microscopy to evaluate the impact of cholesterol lowering drugs on lipid storage in C. elegans . This study shows that statin treatment reduces the level of Nile Red staining, but does not affect neutral lipid storage in C. elegans as detected with CARS microscopy. In a separate study, it was found that Nile Red stains a subset of gut granules, which does not correlate with lipid content ( 72 ). Collectively, these studies suggest that Nile Red staining might not be a reliable measure of lipid content in C. elegans . Most recently, Le et al. ( 48 ) identifi ed coexisting neutral and autofl uorescent lipid species with simultaneous CARS and TPEF imaging ( Fig.  3B ). Fingerprint spectral analysis using confocal Raman microspectroscopy on the same platform provides the for energy, LDs also store phospholipids and sterols, which are critical for the growth and maintenance of the cell membrane ( 51 ). Increased cytoplasmic LD accumulation is proposed to be a mechanism to avoid apoptosis when cells are exposed to excess unesterifi ed fatty acids ( 52 ). The observation of LD interaction with cellular organelles such as the endoplasmic reticulum, mitochondria, and endosomes suggests that LDs might facilitate intracellular lipid transport ( 53 ). Interestingly, LDs might play a role in mediating host-pathogen interaction because LD accumulation increases upon hepatitis C viral infection and the replication of hepatitis C virus interacts with LDs ( 54 ). Most signifi cantly, excess accumulation of cyto plasmic LDs is strongly associated with major diseases including obesity, type II diabetes, hepatic steatosis, and atherosclerosis ( 3 ).
The emerging dynamic view of LDs is attracting biologists to study the biology of LDs. In particular, the discoveries of LD-associated perilipin, adipophilin, and TIP47 (PAT) proteins, Rab18, DGAT2, and caveolin are posing questions about how LDs are formed, grown, and mobilized ( 55 ). More than ever, the ability to monitor LDs in real time is critically needed. With the capability of labelfree visualization of LDs, CARS microscopy is highly suitable for the studies of LD dynamics. Moreover, green fl uorescent protein and red fl uorescent protein-tagged proteins that associate with LDs have been made available ( 56 ). Simultaneous CARS imaging of LDs and TPEF imaging of GFP and RFP-tagged proteins would allow the dynamic correlation between protein localization and LD formation, growth, or mobilization in living cells.
The application of CARS microscopy to the studies of LD biology is slowly gaining traction. Using CARS microscopy, Nan et al. ( 40 ) observed that the formation of large LDs in 3T3-L1 cells was preceded by the initial clearance of small LDs after adipogenesis induction. By simultaneous CARS imaging of LDs and TPEF imaging of labeled mitochondria, Nan et al. ( 57 ) further revealed possible interaction between LDs and cellular organelles in adrenal cortical Y-1 cells. Yamaguchi et al. ( 58 ) used CARS imaging to examine lipolysis of LDs and found a possible role of CGI-58 protein in the vesiculation of small LDs from large LDs. Le et al. ( 59 ) used CARS imaging to examine the phenotypic variability in LDs accumulation among clonal cells and found that variability in LD formation is the consequence of the ability of cells to process insulin. By simultaneous TPEF imaging of viral RNA distribution and CARS imaging of LDs, Lyn et al. ( 60 ) found that lipid metabolism inhibitors can cause disruption of hepatitis C viral replication. These initial studies showcase possible applications of CARS microscopy. However, the full impact of CARS microscopy on LD biology is still dependent on further in-depth studies and broader utilization of CARS imaging capability.

Study of lipid metabolism in C. elegans
The pathways of lipid metabolism are highly conserved from C. elegans to human ( 61,62 ). Being optically transparent and having highly tractable genetics, C. elegans has Third, a plaque can also be identifi ed by CARS visualization of lipid-rich foam cell and extracellular lipid deposits. Hence, Le et al. suggest that multimodal imaging can signifi cantly improve the sensitivity and accuracy of plaque detection. In a different study, Wang  However, clinical application of CARS imaging for early detection of plaques is dependent on the successful development of CARS endoscopy. Currently, the tissue penetration depth of CARS microscopy is c.a. 100 microns. This penetration depth is insuffi cient for visualization of arterial lumen from the adventitia. In addition, connective and fatty tissues surrounding the artery would scatter light and reduce the visibility of CARS imaging. Therefore, effective deployment of CARS imaging should be done from the arterial lumen. This deployment requires CARS endoscopy, where the delivery of the excitation laser and collection of emitted signal are accomplished using fl exible fi ber optics, miniaturized objective, and scan heads ( 84,85 ). Ideally, CARS endoscopy should be deployed on a catheter bundle that is also capable of optical coherent tomography and IVUS ( 75 ). This combinatorial scheme would allow both anatomical mapping of plaques with optical coherent tomography and IVUS and compositional analysis with CARS/SHG/TPEF. Although CARS endoscopy has yet to be realized, recent advances in NLO endoscopy ( 86 ) suggest its eventual reality. The potential benefi ts of CARS endoscopy for the detection and diagnosis of atherosclerotic plaques should encourage future research and development on this front.

CARS imaging of intestinal lipid absorption
Obesity is an established risk factor for major human diseases including cancer, diabetes, and cardiovascular diseases ( 6 ). The prevalence of obesity in developed countries poses a signifi cant challenge to global health management. Obesity is the consequence of the imbalance between energy intake and expenditure. To manage obesity, several approaches have been taken including diet modifi cation, exercise regiment, bariatric surgery ( 87 ), and intestinal sleeve implantation ( 88 ). Although diet and exercise are the most widely accepted recommendation to combat obesity, many people still struggle to reduce their body weight with this approach. Alternatively, bariatric surgery and intestinal sleeve implantation have shown capability of detecting lipid chain unsaturation. Le et al. also measured lipid-chain unsaturation of single LDs in C. elegans with genetic deletions of genes encoding for ⌬ 9 desaturases ( Fig. 3B ). These results highlight the capability of CARS imaging combined with spontaneous Raman microspectroscopy for analysis of complex genotype-phenotype relationships between lipid storage, peroxidation, and desaturation in single living C. elegans . Together, these initial studies showcase the versatility of CARS microscopy for noninvasive and label-free visualization and compositional analysis of LDs in C. elegans . When combined with recent advances in C. elegans genetics ( 73 ) and highthroughput screening ( 74 ), CARS microscopy is expected to facilitate rapid functional studies of lipid metabolism at the genomic scale and advance our understanding of the roles of lipids in human health and diseases.

Detection of atherosclerosis by CARS-based multimodal microscopy
Atherosclerosis is a complex process that begins in early adolescence and progressively develops over time. Nonetheless, the fi rst diagnosis of atherosclerotic plaques is normally when a heart attack occurs. Existing diagnostic tools for atherosclerosis are limited and inadequate for early detection. Noninvasive testing of atherosclerosis such as the cardiac stress test yields apositive diagnosis only when the lumen narrowing is at 75% or greater. Anatomical diagnosis including computed tomography, magnetic resonance imaging, and intravenous ultrasound (IVUS) are expensive and not widely prescribed and can detect only advanced calcifi ed plaques ( 75 ). In addition, complications from atherosclerosis are not only dependent on stenosis or lumen occlusion but also on the rupture of vulnerable plaques. Thus, a diagnosis of atherosclerotic plaques should include both anatomical features for stenosis evaluation and molecular composition for vulnerability evaluation ( 76,77 ). However, the capability of existing diagnostic tools for atherosclerosis is insufficient to satisfy this requirement.
Several recent studies highlight the capability of CARSbased multimodal NLO microscopy for plaque detection and molecular composition analysis. Le et al. ( 78) and Wang et al. ( 79 ) employed multimodal NLO microscopy to visualize the composition of arterial walls and atherosclerotic plaques. They demonstrated label-free visualization of elastin, collagen fi brils, smooth muscle cells, endothelial cells, foam cells, and extracellular lipid deposits using combined TPEF, SHG/SFG, and CARS imaging modalities. Le et al. showed that a plaque can be detected with multimodal NLO imaging by several ways. First, SHG imaging revealed that the arrangement of collagen fi brils is parallel with each other in healthy arterial walls but disorganized in plaques. Second, measured from the lumen, TPEF signal from elastin appears before SHG signal of collagen fi brils in healthy arterial walls, whereas in the plaque areas, SHG signal appears together with TPEF signal because collagen fi brils breach the internal elastic lamina and grow into the lumen. Thus, the order of appearance of TPEF and SHG signal can indicate the plaque location. had improved insulin sensitivity and decreased tissue levels of triacylglycerol compared with wild-type mice. Thus, DGAT-1 was suggested to be a pharmaceutical target for the treatment of human obesity and diabetes ( 91 ). However, the mechanism underlying the protective effect of DGAT-1 was not clearly understood. To study to roles of DAGT-1, Lee et al. ( 92 )

restored the expression of DGAT-1 protein only in the intestine ( Dgat1 IntONLY ) of Dgat1
Ϫ / Ϫ mice. Dgat1 IntONLY mice were not resistant to high-fat dietinduced hepatic steatosis or obesity despite the absence of DGAT-1 protein in the liver and adipose tissues. This study suggests a role of DGAT-1 in regulating dietary fat secretion out of enterocytes, thus affecting the level of dietary fat in tissues. Together, these initial studies demonstrate the use of CARS imaging to study the role of the DGAT-1 enzyme in intestinal lipid absorption. Future applications of CARS imaging should allow functional studies of other enzymes in intestinal lipid absorption or evaluation of drugs aiming at interfering with the energy intake to treat obesity and diabetes.

Study of lipids in cancer development
Intracellular lipid bodies have been observed in many types of cancers ( 93,94 ). Early clinical studies in the 1970s linked lipid-rich carcinoma of mammary glands to high incidence of cancer mortality, metastatic tumors, and aggressive clinical behaviors ( 93,95 ). More recently, increased expression of lipid metabolism genes are found in aggressive brain, mammary, and prostate cancer ( 96,97 ). These observations suggest a link between lipid metabolism and cancer aggressiveness. Nonetheless, lipid metabolism has not been used as a prognosis factor for cancer aggressiveness due to the lack of a mechanistic understanding.
Using CARS microscopy, Le et al. ( 98 ) examined the impacts of excess lipids on cancer development. In a Balb/c mice lung cancer model, Le et al. reported that excess lipids due to diet-induced hyperlipidemia and increased visceral adiposity strongly correlate with increased cancer metastasis. In tissue cultures, Le et al. found that excess lipids caused perturbations to cancer cell membrane, induced cytoplasmic lipid accumulation, and promoted cancer invasiveness. The presence of cytoplasmic lipid bodies allowed CARS visualization of migrating cancer cells on collagen fi bers and circulating tumor cells in blood samples. In recent years, CARS fl ow cytometry has been successfully applied to characterize lipid-rich cells and particles in microfl uidic chambers ( 99,100 ). The observation of lipid-rich circulating tumor cells suggests possible development of label-free intravital CARS fl ow cytometry for clinical cancer diagnosis.
In addition to the direct impacts on cancer cells, Le et al. ( 101 ) reported perturbation by excess lipids on tumor microenvironments. Using a Sprague-Dawley rat mammary cancer model, Le et al. examined the composition of the mammary tumor microenvironment and found larger lipid droplets of adipocytes and higher density of collagen fi brils in diet-induced obese rats as compared with lean rats. Given the known roles of adipocytes in supporting tumor growth ( 102 ) and collagen fi brils in supporting signifi cant reduction of body weight and improvement in the glycemic index. Nonetheless, the long-term effects of surgery and implantation have yet to be determined. Another approach to combat obesity is through pharmaceutical intervention. However, this approach requires in-depth understanding of an individual's metabolic activity, energy homeostasis, genetics, molecular control of adipogenesis, neuronal control of satiety, and other characteristics to design effective drugs with minimal side effects.
An effective approach to restoring the energy balance is to minimize the energy intake by controlling the absorption of dietary lipids ( 89 ). Toward this goal, researchers have studied key aspects of lipid emulsifi cation, hydrolysis, uptake into enterocytes, intracellular traffi cking, storage, and secretion into the circulation. However, tracking the movement of dietary lipids relies on histology and electron microscopy for visualization and bulk measurements for quantitative lipid analysis. These methods provide static snapshots of intestinal lipid absorption but leave the dynamic process largely to extrapolation.
Recently, Zhu et al. ( 90 ) described the fi rst use of CARS microscopy for real-time visualization of lipid absorption by the small intestine in living mice. With minimally invasive surgery, Zhu et al. monitored the enterocytes as a function of time after feeding and found a dynamic cytoplasmic triacylglycerol pool ( Fig. 4A ). Especially, Zhu et al. found that acyl CoA:diacylglycerol acyltransferase 1 (DGAT-1) defi cient mice exhibited higher level of cytoplasmic triacylglycerol storage compared with wild-type mice. Previous studies showed that DGAT-1 defi cient ( Dgat1 Ϫ / Ϫ ) mice were resistant to diet-induced obesity and  ( 90,112 ). mainly aim at inhibiting infl ammation to curtail further attack on the myelin sheaths.
CARS microscopy has been applied successfully to the studies of demyelination in the past several years. The myelin membranes contain about 70% lipid by weight and the high-density CH 2 groups produce a large CARS signal ( 110 ). Wang et al. ( 41 ) reported the fi rst application of CARS microscopy to visualize the myelin sheaths, the node of Ranvier, and the Schmidt-Lanterman incisure in explanted spinal cord tissues. Fu et al. ( 111 )  CARS microscopy is uniquely advantageous for the studies of demyelination for several reasons. First, CARS imaging does not require sample processing to visualize myelin structures as in histology or electron microscopy. This advantage reduces any associated preparation time for samples. Second, CARS imaging does not require labeling for visualization; thus, it allows long-term visualization of the myelin sheaths ( 41,115 ). Third, CARS-based multimodal microscopy allows simultaneous visualization of myelin sheaths and cellular processes during demyelination (111)(112)(113). This capability is invaluable to the studies of molecular mechanisms underlying myelin diseases. Fourth, CARS microscopy allows in vivo imaging of the myelin sheaths ( 43,44,113 ). This capability is invaluable for the investigation of the causes of myelin diseases or the evaluation of drugs aiming at curtailing further demyelination or restoring the structure and function of the myelin sheaths. Given the demonstrated advantages, it is conceivable that CARS microscopy continues to be a technique of choice for the future studies of the myelin diseases.

DISCUSSION
CARS microscopy is a versatile instrument suitable for the studies of lipids in diseases. CARS microscopy relies on the intrinsic molecular vibration for contrast mechanism ( 18 ). Therefore, it is particularly sensitive to the lipid-rich structures due to the abundance of the CH 2 group and the distinctive CH 2 stretch-vibration frequency at 2840 cm The strong signal-to-noise ratio arising from the CH 2 vibration allows for short integration time and enables image acquisition with speed as high as video rate ( 21 ). Continuous applications of CARS microscopy to the studies of lipid-related diseases are shedding light on the full capability of CARS imaging. As lipid-related diseases continue cancer cell migration ( 103,104 ), this observation suggests that excess lipids promote a tumor microenvironment conducive to tumor growth and metastasis.
To satisfy the high proliferation rate, a cancer cell needs to increase its biomass by increasing its uptake of nutrients including glucose and lipids ( 105,106 ). However, as many lipid molecules participate in cell signaling ( 4 ), it is conceivable that they also infl uence cancer cell behaviors. Future studies that focus on profi ling the lipid composition and the behavior of cancer cells could lead to a better understanding of the roles of lipids in cancer. With the capability for label-free visualization and noninvasive compositional analysis of lipids, CARS microscopy is an invaluable tool to the studies of lipids in cancer development.

Study of skin biology
Skin plays an important role in human physiology by providing a protective barrier against germs, an insulation layer against fl uctuating temperatures, and a sensory organ for heat, touch, and pain ( 107 ). Skin consists of three main layers: an epidermis outer layer with melanocytes, a dermis second layer with nerve endings, sweat glands, sebaceous glands, and hair follicles, and a third fatty layer of subcutaneous tissues. Although the skin conditions and diseases are vast, the widely known include melanoma, acne, and hair loss.
Skin is highly accessible to optical examination by being the superfi cial structure. Comprising lipid-rich structures including the sebaceous glands and adipocytes, skin is a suitable target for CARS imaging. Using video-rate CARS microscopy, Evans et al. ( 21 ) described the skin composition in the ears of living mice and tracked the diffusion of topically applied baby oil into the skin. Using compound Raman microscopy, Slipchenko et al. ( 47 ) measured the lipid composition of subcutaneous adipocytes and sebaceous glands. Using CARS microscopy, Zimmerly et al. ( 108 ) measured the distribution and concentration of d-glycine in human hair. Together, these studies highlight a growing interest in the application of CARS microscopy to the studies of skin biology. It is feasible that CARS microscopy will be widely used to track the diffusion of topically applied ointments and drugs and to evaluate the effects of cosmetic, skin care, and hair care products on the texture, moisture, and lipid composition of the skin and hair.

Study of myelin diseases
Demyelination, or the loss of the myelin sheaths around axons, is a hallmark of many neurodegenerative diseases such as leukodystrophies and multiple sclerosis ( 2 ). The loss of the myelin sheaths impairs signal conduction along axons and reduces the communication among nerve cells. Individuals with demyelinated diseases could exhibit any neurological symptom including impairments of speech, cognition, vision, and coordination. The causes of demyelinated diseases are unknown but the risk factors might include genetic, environmental, and infection ( 2,109 ). Universally, the demyelination process involves attacks on the myelin sheaths by the immune system. Currently, there is no cure for demyelinated diseases. Existing treatments of the input laser beams. The heterodyne detection also renders the SRS signal linearly proportional of the vibrational oscillators in the focal volume, making it more sensitive for detection of low-concentration molecules. Importantly, because the SRS process occurs simultaneously with CARS, the SRS modality can be implemented on a CARS microscope with the addition of a laser intensity modulator, a photodiode detector, and a lock-in amplifi er. Although a detailed description of this technique is beyond the scope of this review, two recent applications have shown great potential of SRS microscopy in highspeed vibrational imaging of samples with fi ngerprint Raman bands ( 129,130 ).
to be the leading causes of death worldwide, it is foreseeable that CARS microscopy will emerge as an indispensible tool to illuminate the roles of lipids in health and diseases.
Technically, coupling CARS microscopy with other NLO imaging modalities, such as TPEF and SHG, expands the observable biological structures and broadens the applicability of CARS microscopy to the studies of complex biological systems ( 116 ). Furthermore, the integration of spontaneous Raman spectroscopy onto the CARS microscope platform provides complete high-resolution Raman spectral information of the samples ( 47 ). Compact laser sources ( 38,117,118 ) are being developed to make CARS microscopy more accessible to biomedical researchers by reducing its cost, footprint, and complexity. Increasing the penetration depth with CARS endoscopy ( 84,85 ) or miniature microscope objective ( 119 ) could allow CARS imaging from the lumen of the intestine, esophagus, and artery, or CARS imaging of lipid-rich tissues with minimally invasive surgery. Finally, though CARS microscopes described in recent literature have been home-built, turnkey commercial CARS microscopes have been recently released. The commercialization of CARS microscopy is expected to render this technology more accessible and enable its widespread applications to biomedical research. Collectively, an affordable, easy-to-operate, versatile, and multifunctional CARS microscope promises to be a powerful tool for understanding the role of lipids in health and diseases.
Finally, we discuss limitations of the technique and potential solutions. A major drawback of CARS microscopy is the existence of a nonresonant background. The CARS signal contains a Raman-shift dependent resonant contribution and a Raman-shift independent electronic contribution that could be enhanced in the presence of two-photon electronic resonance. The electronic contribution disperses the CARS spectral profi le ( 120 ) and gives a vibrationally nonresonant background in CARS images. For myelin sheaths and lipid bodies, the high-density C-H vibrational oscillators produce a resonant CARS signal that is much larger than the nonresonant contribution from water and other biological structures. In these cases, the large resonant signals permit selective CARS imaging of myelin in a complex tissue environment and lipid droplets in mammalian cells, model organisms, and mice, as reviewed here. The nonresonant background, however, makes it diffi cult to perform CARS imaging based on the fi ngerprint bands. Various advanced CARS imaging techniques have been developed to suppress the nonresonant contribution, including polarization-sensitive detection ( 35 ), time-resolved detection ( 121 ), heterodyne detection ( 122,123 ), and frequency modulation ( 122,124 ). However, the complexity of these techniques hinders their biological applications.
The recently developed stimulated Raman scattering (SRS) microscopy ( 125-128 ) provides a simple and robust way to overcome the nonresonant background problem. The SRS signal is free of the nonresonant background via heterodyne detection of Raman-induced intensity changes