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)     

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.     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

Intracellular imaging of host‐pathogen interactions using combined CARS and two‐photon fluorescence microscopies

Intracellular imaging is a key tool in the investigation of host‐pathogen interactions. Advances in this area are particularly sought to understand the effect of viral infection processes on the host cell and its metabolic functions including those cases where host cell lipid metabolism is modulated as a result of infection. We demonstrate the use of combined coherent anti‐Stokes Raman scattering (CARS) and two‐photon fluorescence microscopies to image fibroblast cells infected by cytomegalovirus. CARS is used to image the host cell membrane, lipid droplets and morphology of the nucleus. Cell nuclei are found to expand during infection, approximately doubling in area. Some cells also show accumulations of lipid droplets at the nuclear periphery. Using a genetically modified virus strain expressing the green fluorescent protein also enables two‐photon imaging of the same cells to reveal the location, nature and extent of viral protein expression. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Assessing the efficacy of coherent anti‐Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples

Coherent anti‐Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two‐photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5‐aminolevulinic acid‐induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition.

Experimental glioblastoma expressing green fluorescent protein.     

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.     

A comparative Raman and CARS imaging study of colon tissue

An experimental evaluation of the information content of two complimentary techniques, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy, is presented. CARS is a nonlinear variant of Raman spectroscopy that enables rapid acquisition of images within seconds in combination with laser scanning microscopes. CARS images were recorded from thin colon tissue sections at 2850, 1660, 1450 and 1000 cm^–1 and compared with Raman images. Raman images were obtained from univariate and multivariate (k‐means clustering) methods, whereas all CARS images represent univariate results. Variances within tissue sections could be visualized in chemical maps of CARS and Raman images. However, identification of tissue types and characterization of variances between different tissue sections were only possible by analysis of cluster mean spectra, obtained from k‐means cluster analysis. This first comparison establishes the foundation for further development of the CARS technology to assess tissue. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.     

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.      

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.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

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.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

The non‐destructive and label‐free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non‐contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400–1800 cm^–1), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof‐of‐principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically‐degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non‐destructive and non‐contact tool to probe ECM state in situ. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2‐photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre‐resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization‐resolved SHG signals. Furthermore, 2‐photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.      

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

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.     

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti‐Stokes Raman scattering (CARS), third‐order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white‐light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study.

Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in‐plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.