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.     

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.     

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.     

Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.     

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.     

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.     

Label-free imaging of lipid dynamics using Coherent Anti-stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy

The recently developed Coherent Anti-stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy have provided new methods to visualize the localization and regulation of biological molecules without the use of invasive and potentially perturbative labels. They allow rapid imaging of specific molecules with high resolution and sensitivity. These tools have been effectively applied to the study of lipid metabolism using Caenorhabditis elegans as a genetic model, unraveling new lipid storage phenotypes and their regulatory mechanisms. Here we review the underlying principle of CARS and SRS microscopy, as well as their recent applications in lipid biology research in C. elegans.     

Nonlinear vibrational microscopy applied to lipid biology

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.     

Imaging growth of neurites in conditioned hydrogel by coherent anti-Stokes Raman scattering microscopy

Cultured DRGs in different gel scaffolds were analyzed using CARS microscopy to determine its possible use as a label-free imaging option for tracking cellular growth in a gel scaffold. This study demonstrates for the first time the applicability of CARS microscopy to the imaging of live neuronal cells in GAG hydrogels. By tuning the laser beating frequency, ωp ? ωs, to match the vibration of C-H bonds in the cell membrane, the CARS signal yields detailed, high-quality images of neurites with single membrane detection sensitivity. The results demonstrate that CARS imaging allows monitoring of cellular growth in a tissue scaffold over time, with a contrast that shows comparable cellular structures to those obtained using standard fluorescent staining techniques. These findings show the potential of CARS microscopy to assist in the understanding of organogenesis processes in a tissue scaffold.     

Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.     

Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging

The accumulation of lipids, including cholesterol, in the arterial wall plays a key role in the pathogenesis of atherosclerosis. Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR(-/-)) mouse and the ApoE lipoprotein-deficient (ApoE(-/-)) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE(-/-) mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.     

Coherent anti-stokes Raman scattering microscopy: a biological review.

Microscopic imaging of cells and tissues are generated by the interaction of light with either the sample itself or contrast agents that label the sample. Most contrast agents, however, alter the cell in order to introduce molecular labels, complicating live cell imaging. The interaction of light from multiple laser sources has given rise to microscopy, based on Raman scattering or vibrational resonance, which demonstrates selectivity to specific chemical bonds while imaging unmodified live cells. Here, we discuss the nonlinear optical technique of coherent anti-Stokes Raman scattering (CARS) microscopy, its instrumentation, and its status in live cell imaging.     

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.     

The impact of cell fixation on coherent anti‐stokes Raman scattering signal intensity in neuronal and glial cell lines

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid‐related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti‐stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.      

Signal generation and Raman‐resonant imaging by non‐degenerate four‐wave mixing under tight focusing conditions

The authors demonstrate Raman‐resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3‐color coherent anti‐Stokes Raman scattering (CARS) experiment. The interaction of three coincident short‐pulsed laser beams simultaneously generates both 2‐color (degenerate) CARS and 3‐color (non‐degenerate) CARS signals, which are collected and characterized spectroscopically – allowing for resonant, doubly‐resonant, and non‐resonant contrast mechanisms. Images obtained from both 2‐color and 3‐color CARS signals are compared and found to provide complementary information. The 3‐color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label‐free non‐linear microscopy, e.g. by probing multiple Raman resonances. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

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.     

Interpreting CARS images of tissue within the C–H‐stretching region

Single band coherent anti‐Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video‐rate image acquisition of tissue. This is due to the large Raman signal in the C—H‐stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C—H‐stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C—H‐stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multimodal nonlinear optical imaging of collagen arrays

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.