Coherent Raman Scattering Research Articles

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Coherent Raman scattering microscopy: capable solution in search of a larger audience.

.Significance: Coherent Raman scattering (CRS) microscopy is an optical imaging technique with capabilities that could benefit a broad range of biomedical research studies.Aim: We reflect on the birth, rapid rise, and inescapable growing pains of the technique and look back on nearly four decades of developments to examine where the CRS imaging approach might be headed in the next decade to come.Approach: We provide a brief historical account of CRS microscopy, followed by a discussion of the challenges to disseminate the technique to a larger audience. We then highlight recent progress in expanding the capabilities of the CRS microscope and assess its current appeal as a practical imaging tool.Results: New developments in Raman tagging have improved the specificity and sensitivity of the CRS technique. In addition, technical advances have led to CRS microscopes that can capture hyperspectral data cubes at practical acquisition times. These improvements have broadened the application space of the technique.Conclusion: The technical performance of the CRS microscope has improved dramatically since its inception, but these advances have not yet translated into a substantial user base beyond a strong core of enthusiasts. Nonetheless, new developments are poised to move the unique capabilities of the technique into the hands of more users.

Dissecting lipid droplet biology with coherent Raman scattering microscopy.

Lipid droplets (LDs) are lipid-rich organelles universally found in most cells. They serve as a key energy reservoir, actively participate in signal transduction and dynamically communicate with other organelles. LD dysfunction has been associated with a variety of diseases. The content level, composition and mobility of LDs are crucial for their physiological and pathological functions, and these different parameters of LDs are subject to regulation by genetic factors and environmental inputs. Coherent Raman scattering (CRS) microscopy utilizes optical nonlinear processes to probe the intrinsic chemical bond vibration, offering label-free, quantitative imaging of lipids in vivo with high chemical specificity and spatiotemporal resolution. In this Review, we provide an overview over the principle of CRS microscopy and its application in tracking different parameters of LDs in live cells and organisms. We also discuss the use of CRS microscopy in genetic screens to discover lipid regulatory mechanisms and in understanding disease-related lipid pathology.

A label-free, fast and high-specificity technique for plant cell wall imaging and composition analysis

BackgroundNew cell wall imaging tools permit direct visualization of the molecular architecture of cell walls and provide detailed chemical information on wall polymers, which will aid efforts to use these polymers in multiple applications; however, detailed imaging and quantification of the native composition and architecture in the cell wall remains challenging.ResultsHere, we describe a label-free imaging technology, coherent Raman scattering (CRS) microscopy, including coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy, which can be used to visualize the major structures and chemical composition of plant cell walls. We outline the major steps of the procedure, including sample preparation, setting the mapping parameters, analysis of spectral data, and image generation. Applying this rapid approach will help researchers understand the highly heterogeneous structures and organization of plant cell walls.ConclusionsThis method can potentially be incorporated into label-free microanalyses of plant cell wall chemical composition based on the in situ vibrations of molecules.

Coherent Raman Scattering Microscopy for Quantitative Label-Free Bioimaging

Coherent Raman scattering (CRS) microscopy is a powerful technique for imaging living cells and tissues label-free, with high chemical specificity and 3D spatial resolution. We have developed a broadband coherent anti-Stokes Raman scattering (CARS) instrument based on a single 5fs Ti:Sa laser source, capable of exciting a wide vibrational range from 1000cm−1 to 3500cm−1 which allows us to perform hyperspectral microscopy [1]. This in turn has led to the development of quantitative chemical imaging algorithms to represent the hyperspectral dataset as a superposition of Raman spectra and concentration maps of individual chemical components (e.g.

Raman Imaging: An Impending Approach Towards Cancer Diagnosis.

In accordance with the recent studies, Raman spectroscopy is well experimented as a highly sensitive analytical and imaging technique in biomedical research, mainly for various disease diagnosis including cancer. In comparison with other imaging modalities, Raman spectroscopy facilitate numerous assistances owing to its low background signal, immense spatial resolution, high chemical specificity, multiplexing capability, excellent photo stability and non-invasive detection capability. In cancer diagnosis Raman imaging intervened as a promising investigative tool to provide molecular level information to differentiate the cancerous vs non-cancerous cells, tissues and even in body fluids. Anciently, spontaneous Raman scattering is very feeble due to its low signal intensity and long acquisition time but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues. So, the present review focuses on the recent developments and applications of Raman spectroscopy-based imaging techniques for cancer diagnosis.

Quantitative Label-Free Imaging of Lipid Domains in Single Bilayers by Hyperspectral Coherent Raman Scattering.

Lipid phase separation in cellular membranes is thought to play an important role in many biological functions. This has prompted the development of synthetic membranes to study lipid–lipid interactions in vitro, alongside optical microscopy techniques aimed at directly visualizing phase partitioning. In this context, there is a need to overcome the limitations of fluorescence microscopy, where added fluorophores can significantly perturb lipid packing. Raman-based optical imaging is a promising analytical tool for label-free chemically specific microscopy of lipid bilayers. In this work, we demonstrate the application of hyperspectral coherent Raman scattering microscopy combined with a quantitative unsupervised data analysis methodology developed in-house to visualize lipid partitioning in single planar membrane bilayers exhibiting liquid-ordered and liquid-disordered domains. Two home-built instruments were utilized, featuring coherent anti-Stokes Raman scattering and stimulated Raman scattering modalities. Ternary mixtures of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol were used to form phase-separated domains. We show that domains are consistently resolved, both chemically and spatially, in a completely label-free manner. Quantitative Raman susceptibility spectra of the domains are provided alongside their spatially resolved concentration maps.

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging.

A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

Mammalian cell and tissue imaging using Raman and coherent Raman microscopy.

Direct imaging of metabolism in cells or multicellular organisms is important for understanding many biological processes. Raman scattering (RS) microscopy, particularly, coherent Raman scattering (CRS) such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has emerged as a powerful platform for cellular imaging due to its high chemical selectivity, sensitivity, and imaging speed. RS microscopy has been extensively used for the identification of subcellular structures, metabolic observation, and phenotypic characterization. Conjugating RS modalities with other techniques such as fluorescence or infrared (IR) spectroscopy, flow cytometry, and RNA-sequencing can further extend the applications of RS imaging in microbiology, system biology, neurology, tumor biology and more. Here we overview RS modalities and techniques for mammalian cell and tissue imaging, with a focus on the advances and applications of CARS and SRS microscopy, for a better understanding of the metabolism and dynamics of lipids, protein, glucose, and nucleic acids in mammalian cells and tissues. This article is categorized under: Laboratory Methods and Technologies > Imaging Biological Mechanisms > Metabolism Analytical and Computational Methods > Analytical Methods.

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

High-contrast, fast chemical imaging by coherent Raman scattering using a self-synchronized two-colour fibre laser

Coherent Raman scattering (CRS) microscopy is widely recognized as a powerful tool for tackling biomedical problems based on its chemically specific label-free contrast, high spatial and spectral resolution, and high sensitivity. However, the clinical translation of CRS imaging technologies has long been hindered by traditional solid-state lasers with environmentally sensitive operations and large footprints. Ultrafast fibre lasers can potentially overcome these shortcomings but have not yet been fully exploited for CRS imaging, as previous implementations have suffered from high intensity noise, a narrow tuning range and low power, resulting in low image qualities and slow imaging speeds. Here, we present a novel high-power self-synchronized two-colour pulsed fibre laser that achieves excellent performance in terms of intensity stability (improved by 50 dB), timing jitter (24.3 fs), average power fluctuation (<0.5%), modulation depth (>20 dB) and pulse width variation (<1.8%) over an extended wavenumber range (2700–3550 cm−1). The versatility of the laser source enables, for the first time, high-contrast, fast CRS imaging without complicated noise reduction via balanced detection schemes. These capabilities are demonstrated in this work by imaging a wide range of species such as living human cells and mouse arterial tissues and performing multimodal nonlinear imaging of mouse tail, kidney and brain tissue sections by utilizing second-harmonic generation and two-photon excited fluorescence, which provides multiple optical contrast mechanisms simultaneously and maximizes the gathered information content for biological visualization and medical diagnosis. This work also establishes a general scenario for remodelling existing lasers into synchronized two-colour lasers and thus promotes a wider popularization and application of CRS imaging technologies.

相干拉曼散射显微技术及其在生物医学领域的应用

相干拉曼散射显微技术及其在生物医学领域的应用

Coherent Raman Scattering Unravelling Mechanisms Underlying Skull Optical Clearing for Through-Skull Brain Imaging.

Optical access of a mouse brain using microscopes is the key to study brain structures and functions in vivo. However, the opaque skull of a mouse has to be either opened or thinned in an invasive way to attain an adequate imaging depth in the brain. Mild skull optical clearing is highly desired, but its chemical mechanism is far from being understood. Here, we unraveled the molecular process underlying optical clearing of the mouse skull by label-free hyperspectral stimulated Raman scattering (SRS) microscopy, thereby discovering the optimal clearing strategy to turn a turbid skull into a transparent skull window. Furthermore, we demonstrated in vivo three-photon imaging of vascular structures as deep as 850 μm in the cortex of the mouse brain. Coherent Raman based microspectroscopy holds great promise to advance skull and tissue clearing methods in the future.

Background-free time-resolved coherent Raman spectroscopy (CSRS and CARS): Heterodyne detection of low-energy vibrations and identification of excited-state contributions

Coherent Raman scattering (CRS) spectroscopy techniques have been widely developed and optimized for different applications in biomedicine and fundamental science. The most utilized CRS technique has been coherent anti-Stokes Raman scattering (CARS), and more recently, stimulated Raman scattering. Coherent Stokes Raman scattering (CSRS) has been largely ignored mainly because it is often strongly affected by fluorescence, particularly for resonance enhanced measurements. However, in the cases of resonant excitation, the information contained in the CSRS signal can be different and complementary to that of CARS. Here, we combine the approaches of pulse shaping, interferometric heterodyne detection, 8-step phase cycling, and Fourier-transform of time-domain measurements, developed in CARS and 2D electronic spectroscopy communities, to measure resonant CSRS and CARS spectra using a titanium:sapphire oscillator. The signal is essentially background-free (both fluorescent and nonresonant background signals are suppressed) with high spectral resolution and high sensitivity and can access low-energy modes down to ∼30 cm−1. We demonstrate the ability to easily select between CSRS and CARS schemes and show an example in which acquisition of both CSRS and CARS spectra allows vibrational modes on the excited electronic state to be distinguished from those on the ground electronic state.

Quantitative imaging of lipid droplets in single cells.

The combination of next generation sequencing (NGS) and automated liquid handling platforms has led to a revolution in single-cell genomic studies. However, many molecules that are critical to understanding the functional roles of cells in a complex tissue or organs, are not directly encoded in the genome, and therefore cannot be profiled with NGS. Lipids, for example, play a critical role in many metabolic processes but cannot be detected by sequencing. Recent developments in quantitative imaging, particularly coherent Raman scattering (CRS) techniques, have produced a suite of tools for studying lipid content in single cells. This article reviews CRS imaging and computational image processing techniques for non-destructive profiling of dynamic changes in lipid composition and spatial distribution at the single-cell level. As quantitative CRS imaging progresses synergistically with microfluidic and microscopic platforms for single-cell genomic analysis, we anticipate that these techniques will bring researchers closer towards combined lipidomic and genomic analysis.

光纤式相干拉曼散射成像光源研究进展

光纤式相干拉曼散射成像光源研究进展

Characterizing stratum corneum structure, barrier function, and chemical content of human skin with coherent Raman scattering imaging.

The most superficial layer of the epidermis, the stratum corneum, plays a crucial role in retaining hydration; if its structure or composition is compromised, dry skin may result as a consequence of poor water retention. Dry skin is typically treated with topical application of humectant agents that attract water into the skin. Corneometry, the industry standard for measuring skin hydration, works by assessing the bulk electrical properties of skin. However, this technique samples a large volume of tissue and thus does not resolve the biochemical changes that occur at the cellular level that may underlie mechanisms of dry skin. These limitations can be addressed using coherent Raman scattering (CRS) microscopy to probe the intrinsic vibrational modes of chemical groups such as lipids and water. In the present study, ex vivo human skin explants undergoing dehydration and humectant-induced rehydration were measured via CRS imaging and corneometry. Corneometry data and chemically specific images were obtained from the stratum corneum of each patient sample at each timepoint. The resulting data was statistically analyzed using linear mixed effect model regression analysis. The cellular imaging data revealed water loss in the stratum corneum during dehydration that was correlated with corneometer readings. Interestingly, the imaging data and corneometer readings show differences under the experimental rehydration conditions. The rehydration results suggest that hydration restored by the humectant agents may not be retained by the corneocytes in the ex vivo model system. Given the complementary nature of corneometry, a bulk assessment tool, and CRS microscopy, a modality with subcellular resolution implemented here in an en-face tissue imaging setup, these techniques can be used to measure uptake and efficacy of topical compounds in order to better understand their mode of action and improve therapeutic applications.

Pulse length variation causing spectral distortions in OPO-based hyperspectral coherent Raman scattering microscopy

Picosecond optical parametric oscillators (OPOs) with broad wavelength tunability are frequently used as light sources in hyperspectral coherent Raman scattering (CRS) microscopy. We investigate how changes in the pulse length during OPO wavelength tuning of the pump beam affect hyperspectral CRS imaging. We find that significant distortions of the resulting CRS spectra occur if the OPO is operated without monitoring pulse length variations. By utilizing a custom-written MATLAB based control program to counteract changes in pulse length, normalized and reproducible data sets can be acquired. We demonstrate this by comparing hyperspectral data obtained from pure substances, as well as relevant biological specimens.

Broadband Coherent Raman Scattering Microscopy

AbstractSpontaneous Raman (SR) microscopy allows label‐free chemically specific imaging based on the vibrational response of molecules; however, due to the low Raman scattering cross section, it is intrinsically slow. Coherent Raman scattering (CRS) techniques, by coherently exciting vibrational oscillators in the focal volume, increase signal levels by several orders of magnitude under appropriate conditions. In its single‐frequency version, CRS microscopy has reached very high imaging speeds, up to the video rate; however, it provides information which is not sufficient to distinguish spectrally overlapped chemical species within complex heterogeneous systems, such as cells and tissues. Broadband CRS combines the acquisition speed of CRS with the information content of SR, but it is technically very demanding. In this Review, the current state of the art in broadband CRS microscopy, both in the coherent anti‐Stokes Raman scattering (CARS) and the stimulated Raman scattering (SRS) versions are reviewed. Different technical solutions for broadband CARS and SRS, working both in the frequency and in the time domains, are compared and their merits and drawbacks assessed.

Invited Article: Heterodyne dual-polarization epi-detected CARS microscopy for chemical and topographic imaging of interfaces

Coherent Raman Scattering (CRS) has emerged in the last decade as a powerful multiphoton microscopy technique offering chemically specific label-free imaging in real time with high three-dimensional spatial resolution. Many technical realizations of CRS microscopy have been proposed to remove, suppress, or account for the non-resonant background in the nonlinear susceptibility which complicates spectral analysis and reduces image contrast. Here, we demonstrate coherent anti-Stokes Raman scattering microscopy using a dual-polarization balanced heterodyne detection in epi-geometry (eH-CARS), providing background-free chemically specific image contrast for nanoparticles and interfaces, shot-noise limited detection, and phase sensitivity. We show the sensitivity and selectivity of eH-CARS in comparison with forward CARS and stimulated Raman scattering on polystyrene beads in agarose gel. As an important biologically relevant application, we demonstrate eH-CARS imaging of individual lipid bilayers with high contrast and topographic sensitivity.