Coherent anti-Stokes Raman Scattering Microspectroscopy Research Articles

Rapid detection and identification of microplastics from nonchemically treated soil with CARS microspectroscopy

In conventional microplastic (MP) analysis, acid or alkaline digestion is a necessary pretreatment step to remove residual organic matter from environmental samples. However, such a digestion process is not only cumbersome and time-consuming, but also possibly cause severe chemical damage to the MP itself, often making accurate MP characterization difficult. This study demonstrates that broadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy is useful for rapidly detecting and identifying MPs in natural soil without any digestion process. A feasibility test is performed with soil samples, which are known to require the most complicated chemical pretreatment for MP analysis, deliberately mixed with various MP particles. The C–H bond-specific CARS imaging and spectral analysis allow rapid MP particle search and chemical identification even in the presence of other residual particles and strongly fluorescent substances from the soil. It is anticipated that this nondestructive, chemical pretreatment-free CARS approach will be a beneficial tool for studying the ecological impacts of MPs absorbed by terrestrial life, such as plants and soil organisms, as well as for complementary analysis of MPs subject to chemical degradation by digestion in investigating the environmental contamination of the MPs.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Multivariate curve resolution with autoencoders for CARS microspectroscopy

Coherent anti-Stokes Raman scattering (CARS) microspectroscopy is a powerful tool for label-free cell imaging thanks to its ability to acquire a rich amount of information. An important family of operations applied to such data is multivariate curve resolution (MCR). It aims to find main components of a dataset and compute their spectra and concentrations in each pixel. Recently, autoencoders began to be studied to accomplish MCR with dense and convolutional models. However, many questions, like the results variability or the reconstruction metric, remain open and applications are limited to hyperspectral imaging. In this article, we present a nonlinear convolutional encoder combined with a linear decoder to apply MCR to CARS microspectroscopy. We conclude with a study of the result variability induced by the encoder initialization.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

Coherent anti-Stokes Raman scattering microspectroscopy: an emerging technique for non-invasive optical assessment of a local bio-nano-environment.

Raman spectroscopy provides a non-invasive, chemically-specific optical imaging of biological objects without relying on endogenous labels. Nonlinear Raman spectroscopy allows non-invasive imaging at much faster speed with an improved spatial resolution and axial sectioning capability. In this report we propose a novel use of nonlinear Raman spectroscopy as a sensor of local nano-environment. Time-resolved coherent anti-Stokes Raman spectrograms are found to be sensitive to small variations of local structural changes, which are not normally observed using conventional Raman spectroscopy.

Investigation by CARS microscopy of squalene and boron nitride as a precursor material for drug delivery carrier

Recently organic squalene and inorganic boron nitride have been considered as precursor materials for drug delivery systems. Drug carriers introduced in the organism need to be monitored to control the drug release within the body. Squalene or boron nitride-based prodrugs usually form nanoparticles or micelles which can be monitored by optical microscopy utilizing fluorescence light originating from drug molecules or dye molecules intentionally introduced for imaging purposes. Coherent anti-stokes Raman scattering (CARS) microscopy is an alternative approach to visualize such particles without labeling. This label-free imaging, based on molecular vibrations is a promising technique in cases where the dye staining cannot be used because of its toxicity or bio-incompatibility. In this paper, we present the CARS microspectroscopy study of squalene and boron nitride materials providing their spectra and images within the fingerprint region.

Plaque-associated lipids in Alzheimer's diseased brain tissue visualized by nonlinear microscopy.

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

Red-shifted solitons for coherent anti-Stokes Raman scattering microspectroscopy in a polarization-maintaining photonic crystal fiber

An alternative light source for coherent anti-Stokes Raman scattering (CARS) microspectroscopy based on red-shifted solitons in a polarization-maintaining photonic crystal fiber (PM-PCF) is experimentally demonstrated. By coupling femtosecond pulses into the anomalous dispersion region of the fundamental mode of a PM-PCF along the slow and fast axes, the red-shifted solitons generated can be used as the Stokes beams when the pump pulses are chosen as the pump beams. Through the process of red-shift, the frequency differences of the pump-Stokes beams are tunable in the ranges of 0 to 4068 cm−1 and 0 to 4594 cm−1, respectively. Moreover, because of the well maintained polarization states of the pump and Stokes beams and the high output powers of the solitons, CARS microspectroscopy using the proposed source will have a high signal-to-noise ratio and short data acquisition time. CARS microspectroscopy based on the proposed all-fiber light source can be used for studying a wide range of vibrational Raman spectra.

Imaging of lipids in microalgae with coherent anti-stokes Raman scattering microscopy.

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r(2) = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.

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.

Chemical imaging of lignocellulosic biomass by CARS microscopy

Chemical and structural composition of wood biomass is studied by label-free and chemically specific Coherent Anti-Stokes Raman Scattering (CARS) microscopy. A concept developed for assignment and semi-quantitative imaging of sample components; cellulose, hemicellulose, and lignin; by multiplex CARS microspectroscopy and subsequent data analysis is presented. Specific imaging without fluorescence backround is achieved an order of magnitude faster compared with conventional Raman microscopy. Laser polarization control yield information on molecular arrangement in wood fibers. Narrowband CARS excitation of single vibrations allows for three-dimensional volume imaging. Thus, CARS microscopy has potential as an important instrument for characterization of lignocellulosic materials.

A fiber coupled and stabilized microscope for analytical CARS micro-spectroscopy

This contribution presents an alternative setup for analytical micro-spectroscopy by means of coherent anti-Stokes Raman scattering (CARS). Instead of using separate detectors for forward- and epi-scattered photons, this setup enhances the comparability of the signals by coupling them into a multimodal optical fiber and employing a fiber optical switch for selection of the signal source at a single detector. To facilitate measurements at different spectral positions, which might be hampered by laser power fluctuations, a feedback loop is introduced into the pump beam to ensure stable and pre-defined laser power levels. The scheme of the microscope and the control software is presented. Its potential is evaluated by recording spectral data of model analytes as well as images of human skin.

High-Speed Imaging of Broadband Multiplex Coherent Anti-Stokes Raman Scattering Microscopy Using a Supercontinuum Source

We combined broadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy with CARS microscopy using a femtosecond laser and a photonic crystal fiber. Broadband multiplex CARS microspectroscopy, which simultaneously measures broadband range (including significant Raman vibrational modes) from 500 to 3500 cm-1 at a high spectral resolution was subsequently performed. Also, via multiplex CARS microscopy using a pair of galvanometer mirrors we successfully demonstrated the high-speed imaging of polymer beads and a lily pollen grain for the study of cell biology dynamics. The imaging speed was 37 s per image, which is 30–40 times faster than that previously reported in broadband CARS microscopy.

Molecular Mechanisms of Active Lipolysis in 3T3-L1 Adipocytes

Lipid droplets (LDs) of mammalian cells are coated with lipid-associated proteins (LAP) such as perilipin (PLIN), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL), which play important roles in regulating fat storage and mobilization, by interacting with each other. By using coherent anti-Stokes Raman Scattering (CARS) microscopy which visualizes LDs in living cells, we observed numerous micro-LDs (mLDs) emerging after lipolytic stimulation. However, the interplay of LAP and the physiological significance of mLDs are not fully understood. PURPOSE: In this study, we examined the localization, trafficking, and interactions of key proteins involved in lipolysis (i.e. LAP) as well as the physiological significance of mLDs during lipolysis in differentiated 3T3-L1 adipocytes. METHODS: The localization and trafficking of LAP during 2 hrs of cAMP-dependent PKA activation were examined by immunostaining. In addition, dynamic morphological changes of LDs and fatty acid (FA) turnover during lipolysis were obtained using multiplex CARS microspectroscopy, which is able to distinguish between deuterium-labeled FA and non-labeled FA. Lipolytic activity and the protein-protein interaction were biochemically analyzed. RESULTS: In the basal state, the localization of HSL was cytoplasmic, while that of PLIN was on large LDs. Upon lipolytic stimulation, HSL being redistributed to the surface of large LDs where being co-localized with PLIN. Furthermore, HSL and PLIN were co-localized at particulate structures, which are supposed to be mLDs. We found that these structures are derived from endoplasmic reticulum (ER) where nascent LDs are formed via triacylglycerol (TAG) synthesis, and perform active lipolysis. On the other hand, lipolytic stimulation did not alter the cytoplasmic distribution of ATGL. Following subcellular fractionation, we observed that ATGL and its co-activator CGI-58 were co-fractionated upon lipolytic stimulation in membrane fraction where organelles are enriched. Furthermore, immunoprecipitation analysis demonstrated that a small but significant amount of ATGL was co-precipitated with Myc-tagged CGI-58 in membrane fraction, in response to lipolytic stimulation. CONCLUSIONS: These results suggest that mLDs have important functions to enhance the efficiency of lipolysis, by increasing the total LD surface area where PLIN and HSL can interact. It is also possible that active lipolysis occurs by means of interplays of CGI-58 and ATGL, in organelles other than LDs.

Label-Free Imaging of Lipophilic Bioactive Molecules during Lipid Digestion by Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy

The digestion and absorption of lipophilic, bioactive molecules such as lipids, physiologically active nutrients (nutraceuticals), and drugs play a crucial role in human development and health. These molecules are often delivered in lipid droplets. Currently, the kinetics of digestion of these lipid droplets is followed by in vitro models that simulate gastrointestinal conditions, while phase changes within the lipid droplets are observed by light or electron microscopy. However real-time, spatially resolved information about the local chemical composition and phase behavior inside the oil droplet is not accessible from these approaches. This information is essential as the surface and phase behavior determine the local distribution of molecules in the oil droplets and thus may influence the rate of uptake, for example, by impairing the effective transfer of bioactive molecules to intestinal cells. We demonstrate the capability of multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to image the digestion process non-invasively, with submicrometer resolution, millimolar sensitivity, and without the need for labeling. The lipolysis of glyceryl trioleate emulsion droplets by porcine pancreatic lipase is imaged, and the undigested oil and the crystalline lipolytic products are distinguished by their different vibrational signatures. The digestion of droplets containing the phytosterol analogue ergosterol is also probed, and the crystals are observed to dissolve into the lipolytic products. The lipophilic drug progesterone and Vitamin D(3) are dissolved in glyceryl trioctanoate emulsion droplets, and the local concentration is mapped with millimolar sensitivity. The bioactive molecules are observed to concentrate within the droplets as the oil is hydrolyzed. This observation is ascribed to the low solubility of these molecules in the lipolytic products for this system. Neither the type of bioactive molecule nor the initial radius of the emulsion droplet had a large effect upon the rate of digestion under these conditions; lipolysis of the triglyceride by pancreatic lipase appears insensitive to the type of bioactive molecule in solution. These findings shed important new light on lipid digestion and open new possibilities for the chemical visualization of lipid digestion and phase changes in lipid droplets containing bioactive molecules, which in combination with other existing techniques will provide a full picture of this complex physicochemical process.

Two-photon absorption-induced effects in femtosecond coherent anti-Stokes Raman-scattering microspectroscopy of silicon photonic components

We study the effects related to two-photon absorption (TPA) in the microspectroscopy of the silicon photonic components based on coherent anti-Stokes Raman scattering (CARS) of femtosecond pulses. With 300-fs pulses of 1.24-μm Cr:forsterite laser radiation delivering pump and probe fields and a frequency-shifted soliton output of a large-mode area photonic-crystal fiber employed as a Stokes field, pronounced TPA effects have been observed in the CARS microspectroscopy of silicon components for pump-pulse intensities exceeding 10 GW/cm2.

Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared

Subnanosecond supercontinuum (SC) has been generated by a 1,064 nm microchip laser combined with a photonic crystal fiber. The ultrabroadband (>2,000 cm(-1)) SC has facilitated multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy in the spectral range from 1,000 to 3,000 cm(-1) with lateral and depth spatial resolution of 0.9 and 4.6 microm, respectively. A clear CARS image of a Nicotiana tabacum L. cv. Bright Yellow 2 cell has been obtained with high vibrational contrast.

Microanalytical nonlinear single-beam spectroscopy combining an unamplified femtosecond fibre laser, pulse shaping and interferometry

Nonlinear vibrational spectroscopy using a single beam of femtosecond pulses from an unamplified fibre laser oscillator is demonstrated. To achieve high spectral resolution and sensitive signal detection with the picojoule pulse energies available, pulse shaping and integrated interferometric detection is employed. The spectroscopic technique used is coherent anti-Stokes Raman scattering (CARS), which yields well-resolved spectra of molecular vibrations in the 100–350 cm-1 domain of halomethane samples in the liquid phase. We explore the implications of phase control for the interferometric detection of weak signals. The presented combination of a fiber laser, pulse shaping and CARS microspectroscopy is a first example of simplified schemes for compact and robust nonlinear spectroscopic detection and sensing, which is demonstrated exemplarily by on-line monitoring of the chemical composition in a microfluidic flow cell.

白色レーザーを用いた非線形ラマン分光イメージング

Coherent anti-Stokes Raman scattering (CARS) microscopy and microspectroscopy have become powerful techniques for three-dimensional vibrational imaging of chemical and biological systems. Recently, a significant progress has been made of multiplex CARS microspectroscopy, in which a spectral coverage broader than 3500 cm-1 has been achieved using a white-light laser source. In the present study, we have used a photonic crystal fiber (PCF) to generate a white-light laser and used it as the Stokes light source in the CARS process. Owing to an ultrabroadband spectral profile of the white-light laser, a wide range of the vibrational resonances can be investigated.In the present study, we have performed label-free, multi-color imaging of biological samples using multiplex CARS microspectroscopy.

Coherent anti-Stokes Raman scattering microspectroscopy of silicon components with a photonic-crystal fiber frequency shifter

Coherent anti-Stokes Raman scattering (CARS) microspectroscopy of silicon components is demonstrated with pump and probe fields delivered by a mode-locked Cr:forsterite laser and the frequency-shifted soliton output of a photonic-crystal fiber as a Stokes field. CARS microspectroscopy is shown to allow a visualization of microscale features and defects on the surface of silicon wafers, offering much promise for online diagnostics of electronic and photonic silicon chip components.