Shedding new light on lipid functions with CARS and SRS microscopy

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. This article is part of a Special Issue entitled Tools to study lipid functions.     

Achieving Molecular Selectivity in Imaging Using Multiphoton Raman Spectroscopy Techniques

In the case of most optical imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached fluorophores. Unfortunately, they have significant limitations for live-cell imaging. However, an active Raman method, called Coherent Anti-Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo-induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two-photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.     

Stimulated Raman microscopy (CARS): From principles to applications

A new technique in microscopy is now available which permits to image specific molecular bonds of chemical species present in cells and tissues. The so called Coherent Anti-Stokes Raman Scattering (CARS) approach aims at maximizing the light matter interaction between two laser pulses and an intrinsic molecular vibrational level. This is possible through a non linear process which gives rise to a coherent radiation that is greatly enhanced when the frequency difference between the two laser pulses equals the Raman frequency of the aimed molecular bond. Similar to confocal microscopy, the technique permits to build an image of a molecular density within the sample but doesn't require any labelling or staining since the contrast uses the intrinsic vibrational levels present in the sample. Images of lipids in membranes and tissues have been reported together with their spectral analysis. In the case of very congested media, it is also possible to use a non invasive labelling such as deuterium which shifts the molecular vibration of the C-H bond down to the C-D bond range which falls in a silent region of the cell and tissue vibrational spectra. Such an approach has been used to study lipid phase in artificial membranes. Although the technique is still under development, CARS has now reach a maturity which will permit to bring the technology at a commercial stage in the near future. The last remaining bottleneck is the laser system which needs to be simplified but solutions are now under evaluation. When combined with others more conventional techniques, CARS should give its full potential in imaging unstained samples and like two photons techniques has the potential of performing deep tissues imaging.     

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity ¹. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations ^1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted ³. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals ⁴. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm^-1 CH stretch vibration in lipids and the 2120 cm^-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule''s vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.     

Label-free Evaluation of Hepatic Microvesicular Steatosis with Multimodal Coherent Anti-Stokes Raman Scattering Microscopy

Hepatic microvesicular steatosis is a hallmark of drug-induced hepatotoxicity and early-stage fatty liver disease. Current histopathology techniques are inadequate for the clinical evaluation of hepatic microvesicular steatosis. In this paper, we explore the use of multimodal coherent anti-Stokes Raman scattering (CARS) microscopy for the detection and characterization of hepatic microvesicular steatosis. We show that CARS microscopy is more sensitive than Oil Red O histology for the detection of microvesicular steatosis. Computer-assisted analysis of liver lipid level based on CARS signal intensity is consistent with triglyceride measurement using a standard biochemical assay. Most importantly, in a single measurement procedure on unprocessed and unstained liver tissues, multimodal CARS imaging provides a wealth of critical information including the detection of microvesicular steatosis and quantitation of liver lipid content, number and size of lipid droplets, and lipid unsaturation and packing order of lipid droplets. Such information can only be assessed by multiple different methods on processed and stained liver tissues or tissue extracts using current standard analytical techniques. Multimodal CARS microscopy also permits label-free identification of lipid-rich non-parenchymal cells. In addition, label-free and non-perturbative CARS imaging allow rapid screening of mitochondrial toxins-induced microvesicular steatosis in primary hepatocyte cultures. With its sensitivity and versatility, multimodal CARS microscopy should be a powerful tool for the clinical evaluation of hepatic microvesicular steatosis.     

Automated Identification of Subcellular Organelles by Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.     

Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains

We demonstrate quantitative vibrational imaging of specific lipid molecules in single bilayers using laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with a lateral resolution of 0.25 mum. A lipid is spectrally separated from other molecules by using deuterated acyl chains that provide a large CARS signal from the symmetric CD(2) stretch vibration around 2100 cm(-1). Our temperature control experiments show that d62-DPPC has similar bilayer phase segregation property as DPPC when mixing with DOPC. By using epi-detection and optimizing excitation and detection conditions, we are able to generate a clear vibrational contrast from d62-DPPC of 10% molar fraction in a single bilayer of DPPC/d62-DPPC mixture. We have developed and experimentally verified an image analysis model that can derive the relative molecular concentration from the difference of the two CARS intensities measured at the peak and dip frequencies of a CARS band. With the above strategies, we have measured the molar density of d62-DPPC in the coexisting domains inside the DOPC/d62-DPPC (1:1) supported bilayers incorporated with 0-40% cholesterol. The observed interesting changes of phospholipid organization upon addition of cholesterol to the bilayer are discussed.     

Automated Identification of Subcellular Organelles by Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.     

Label-Free Delineation of Brain Tumors by Coherent Anti-Stokes Raman Scattering Microscopy in an Orthotopic Mouse Model and Human Glioblastoma

Background

Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors.

Methods

Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor''s localization, cell proliferation and vascularization.

Results

The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma.

Conclusions

CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.     

Imaging Lignin-Downregulated Alfalfa Using Coherent Anti-Stokes Raman Scattering Microscopy

Targeted lignin modification in bioenergy crops could potentially improve conversion efficiency of lignocellulosic biomass to biofuels. To better assess the impact of lignin modification on overall cell wall structure, wild-type and lignin-downregulated alfalfa lines were imaged using coherent anti-Stokes Raman scattering (CARS) microscopy. The 1,600-cm^?1 Raman mode was used in CARS imaging to specifically represent the lignin signal in the plant cell walls. The intensities of the CARS signal follow the general trend of lignin contents in cell walls from both wild-type and lignin-downregulated plants. In the downregulated lines, the overall reduction of lignin content agreed with the previously reported chemical composition. However, greater reduction of lignin content in cell corners was observed by CARS imaging, which could account for the enhanced susceptibility to chemical and enzymatic hydrolysis observed previously.     

Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues

We present a vibrational imaging study of axonal myelin under physiological conditions by laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy. We use spinal cord white matter strips that are isolated from guinea pigs and kept alive in oxygen bubbled Krebs' solution. Both forward- and epi-detected CARS are used to probe the parallel axons in the spinal tissue with a high vibrational contrast. With the CARS signal from CH₂ vibration, we have measured the ordering degree and the spectral profile of myelin lipids. Via comparison with the ordering degrees of lipids in myelin figures formed of controlled lipid composition, we show that the majority of the myelin membrane is in the liquid ordered phase. By measuring the myelin thickness and axon diameter, the value of g ratio is determined to be 0.68 with forward- and 0.63 with epi-detected CARS. Detailed structures of the node of Ranvier and Schmidt-Lanterman incisure are resolved. We have also visualized the ordering of water molecules between adjacent bilayers inside the myelin. Our observations provide new insights into myelin organization, complementary to the knowledge from light and electron microscopy studies of fixed and dehydrated tissues. In addition, we have demonstrated simultaneous CARS imaging of myelin and two-photon excitation fluorescence imaging of intra- and extraaxonal Ca²⁺. The current work opens up a new approach to the study of spinal cord injury and demyelinating diseases.     

A Lipid Droplet-Associated GFP Reporter-Based Screen Identifies New Fat Storage Regulators in C.elegans

Fat storage disorders including obesity are pandemic human health problems. As a genetically amenable model organism, Caeno- rhabditis elegans has often been used to explore the molecular mechanisms of fat storage regulation. Dye staining of fixed animals and stimulated Raman scattering （SRS） microscopy methods have been used successfully to study fat storage, but a genetic screening system that takes full advantage of C. elegans transparency to perform live imaging of fluorescent protein reporters has not yet been reported. Here, we investigated the tissue-specific expression of the GFP fusion of Perilipin 1 （PLIN1）, a Drosophila lipid droplet-associated protein, in C. elegans. Our results indicate that PLINI：：GFP labels lipid droplets and can be used as a fat storage indicator in live worms. Through an RNAi screen, we further identified several previously uncharacterized new fat storage regulators.     

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D(2)Q(15)K(2)) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm(-1). CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm(-1)) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm(-1)) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.     

Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy

A new vibrational imaging method based on coherent anti-Stokes Raman scattering (CARS) has been used for high-speed, selective imaging of neutral lipid droplets (LDs) in unstained live fibroblast cells. LDs have a high density of C-H bonds and show a high contrast in laser-scanning CARS images taken at 2,845 cm-1, the frequency for aliphatic C-H vibrations. The contrast from LDs was confirmed by comparing CARS and Oil Red O (ORO)-stained fluorescence images. The fluorescent labeling processes were examined with CARS microscopy. It was found that ORO staining of fixed cells caused aggregation of LDs, whereas fixing with formaldehyde or staining with Nile Red did not affect LDs. CARS microscopy was also used to monitor the 3T3-L1 cell differentiation process, revealing that there was an obvious clearance of LDs at the early stage of differentiation. After that, the cells started to differentiate and reaccumulate LDs in the cytoplasm in a largely unsynchronized manner. Differentiated cells formed small colonies surrounded by undifferentiated cells that were devoid of LDs. These observations demonstrate that CARS microscopy can follow dynamic changes in live cells with chemical selectivity and noninvasiveness. CARS microscopy, in tandem with other techniques, provides exciting possibilities for studying LD dynamics under physiological conditions without perturbation of cell functions.     

Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy

The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism.     

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.     

Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy

Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.     

Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy

Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.     

3D-Printed high-NA catadioptric thin lens for suppression of XPM background in Stimulated Raman Scattering microscopy

Stimulated Raman Scattering (SRS) is a fast chemical imaging technique with remarkable bioscience applications. Cross Phase Modulation (XPM) is a ubiquitous nonlinear phenomenon that can create spurious background signals that render difficult a high-contrast imaging in SRS measurements. The XPM-induced signal is usually suppressed using high numerical aperture (NA) microscope objectives or condensers to collect the transmitted excitation beam. However, these high NA optics feature short working distances, hence they are not compatible with stage-top incubators, that are necessary to perform live-cell time-lapse experiments in controlled environments. Here, we show a 3D printed high NA compact catadioptric lens that fits inside stage-top incubators and allows the collection of XPM-free SRS signals. The lens delivers SRS images and spectra with a quality comparable to a signal collection with a high-NA microscope objective. We also demonstrate the compatibility of the 3D printed lens with other nonlinear microscopies usually associated with SRS in multimodal microscopes.     

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.