Surface Enhanced Nonlinear Raman Processes for Advanced Vibrational Probing.

Surface enhanced Raman scattering (SERS) is not restricted to the well-known one-photon excited spontaneous Raman process that gives information on molecular composition, structure, and interaction through vibrational probing with high sensitivity. The enhancement mainly originates in high local fields, specifically those provided by localized surface plasmon resonances of metal nanostructures. High local fields can particularly support nonlinear Raman scattering, as it depends on the fields to higher powers. By revealing plasmon-molecule interactions, nonlinear Raman processes provide a very sensitive access to the properties of metal nanomaterials and their interfaces with molecules and other materials. This Perspective discusses plasmon-enhanced spontaneous and coherent nonlinear Raman scattering with the aim of identifying advantages that lead to an advanced vibrational characterization of such systems. The discussion will highlight the aspects of vibrational information that can be gained based on specific advantages of different incoherent and coherent Raman scattering and their surface enhancement. While the incoherent process of surface enhanced hyper Raman scattering (SEHRS) gives highly selective and spectral information complementary to SERS, the incoherent process of surface enhanced pumped anti-Stokes Raman scattering (SEPARS) can help to infer effective nonresonant SERS cross sections and allows to see "hot" vibrational transitions. Surface enhanced coherent anti-Stokes Raman scattering (SECARS) and surface enhanced stimulated Raman scattering (SESRS) combine the advantages of high local fields and coherence, which gives rise to high detection sensitivity and offers possibilities to explore molecule-plasmon interactions for a comprehensive characterization of composite and hybrid structures in materials research, catalysis, and nanobiophotonics.

Human brain tissue identification using coherent anti-Stokes Raman scattering spectroscopy and diffuse reflectance spectroscopy for deep brain stimulation surgery.

Significance: We assess the feasibility of using diffuse reflectance spectroscopy (DRS) and coherent anti-Stokes Raman scattering spectroscopy (CARS) as optical tools for human brain tissue identification during deep brain stimulation (DBS) lead insertion, thereby providing a promising avenue for additional real-time neurosurgical guidance. Aim: We developed a system that can acquire CARS and DRS spectra during the DBS surgery procedure to identify the tissue composition along the lead trajectory. Approach: DRS and CARS spectra were acquired using a custom-built optical probe integrated in a commercial DBS lead. The lead was inserted to target three specific regions in each of the brain hemispheres of a human cadaver. Spectra were acquired during the lead insertion at constant position increments. Spectra were analyzed to classify each spectrum as being from white matter (WM) or gray matter (GM). The results were compared with tissue classification performed on histological brain sections. Results: DRS and CARS spectra obtained using the optical probe can identify WM and GM during DBS lead insertion. The tissue composition along the trajectory toward a specific target is unique and can be differentiated by the optical probe. Moreover, the results obtained with principal component analysis suggest that DRS might be able to detect the presence of blood due to the strong optical absorption of hemoglobin. Conclusions: It is possible to use optical measurements from the DBS lead during surgery to identify WM and GM and possibly the presence of blood in human brain tissue. The proposed optical tool could inform the surgeon during the lead placement if the lead has reached the target as planned. Our tool could eventually replace microelectrode recordings, which would streamline the process and reduce surgery time. Further developments are required to fully integrate these tools into standard clinical procedures.

Label-free Raman imaging for screening of anti-inflammatory function food.

Natural bioactive compounds and plant constituents are considered to have a positive anti-inflammatory effect. This study aimed to establish a screening technique for anti-inflammatory function in foods based on label-free Raman imaging. A visible anti-inflammatory analysis method based on coherent anti-Stokes Raman scattering (CARS) was established with an LPS-induced RAW264.7 cell model. Dynamic changes in proteins and lipids were determined at laser pump light wavelengths of 2956 cm-1 and 2856 cm-1, respectively. The method was applied to a plant-based formula (JC) with anti-inflammatory activity. Q-TOF-MS and HPLC analyses revealed the main active constituents of JC as quercetin, kaempferol, l-glutamine, and sodium copper chlorophyllin. In in vitro and in vivo verification experiments, JC showed significant anti-inflammatory activity by regulating the TLR4/NF-κB pathway. In conclusion, this study successfully established a label-free and visible method for screening anti-inflammatory constituents in plant-based food products, which will facilitate the evaluation of functional foods.

A multimodal nonlinear optical microscopy study of the responses of Pseudomonas aeruginosa to blue light and antibiotic treatment.

Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant human pathogen involved in numerous infections. Understanding the response of P. aeruginosa to various treatments is critical to developing new ways for the antimicrobial susceptibly test and more effective treatment methods. Conventional antimicrobial susceptibility tests lack molecular information at the single bacterium level. In this study, we used label-free multimodal nonlinear optical microscopy to identify an autofluorescence signal from pyoverdine, a siderophore of the bacteria, for quantification of P. aeruginosa responses to antibiotics and blue light treatment. We also discovered that the bleaching of the pyoverdine autofluorescence signals is correlated with the inactivation of P. aeruginosa and is perhaps one of the mechanisms involved in the blue light inactivation of P. aeruginosa.

What is cooking in your kitchen: seeing "invisible" with time-resolved coherent anti-Stokes Raman spectroscopy.

Cooking oil is a critical component of human food and its main component, lipid, is influential to health, but assessing its authenticity and quality can be challenging due to its complex chemical composition. In this study, we introduce a novel application of time-resolved coherent anti-Stokes Raman scattering (T-CARS) spectroscopy for detecting adulteration and understanding the mechanisms of lipid oxidation in various cooking oils. Our research surpasses the limitations of conventional spontaneous Raman spectroscopy, demonstrating that intra-molecular interactions from unsaturated bonds in triglycerides significantly influence vibrational dephasing time. We observed that these dephasing times, although diverse initially, converge to a similar value after heating cycles. Notably, a longer vibrational dephasing of the CH2 symmetric stretching mode was found to correlate with a higher lipid oxidation rate. These findings underscore the potential of T-CARS in identifying and characterizing subtle molecular interactions, offering a transformative approach to understanding molecular dynamics. This research paves the way for broader applications of T-CARS across fields such as chemistry, biomedicine, and material science, marking a significant advancement in the development of innovative analytical techniques.

Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.

Near-Infrared Light-Driven Mesoporous SiO2 /Au Nanomotors for Eradication of Pseudomonas aeruginosa Biofilm.

Bacterial biofilms are linked to several diseases and cause resistant and chronic infections in immune-compromised patients. Nanomotors comprise a new field of research showing a great promise within biomedicine but pose challenges in terms of biocompatibility. Nanomotors propelled by thermophoresis could overcome this challenge, as they leave no waste product during propulsion. In this study, mesoporous-silica nanoparticles are coated with a thin layer of gold to make nanomotors, which can be driven by near-infrared (NIR) light irradiation. The prepared mesoporous SiO2 -Au nanomotors exhibit efficient self-propulsion when exposed to NIR irradiation, they penetrate deep through a biofilm matrix, and disperse the biofilm in situ due to the photothermal effect on the Au part of the nanomotors. The velocities of such nanomotors are investigated at different wavelengths and laser powers. Furthermore, the study examines the ability of these nanomotors to eradicate Pseudomonas aeruginosa (P. aeruginosa) biofilm under NIR light irradiation. The conducted study shows that the nanomotor's velocity increases with increasing laser power. The mesoporous SiO2 /Au nanomotors show excellent capabilities to eradicate P. aeruginosa biofilms even under short (30 s-3 min) irradiation time. This study shows great promise for overcoming the challenges related to bacterial biofilm eradication.

Coherent Anti-Stokes Raman Scattering Measurements of Time and Length Scales of Temperature Fluctuations in a Turbulent Flame.

The ability to derive temporal and spatial scales of "instantaneous" local temperature variations in a turbulent flame by means of coherent anti-Stokes Raman scattering (CARS) spectroscopy is demonstrated, for the first time to our knowledge. The measurements employed two CARS spectrometers with synchronized nanosecond pulse-repetitive lasers. The system was enabling to record, with a high temporal resolution of about 10 ns, series of single laser shot CARS spectra of N2 molecules from two spatially overlapped or displaced probe volumes as small as 0.03 × 0.03 × 2 mm3. The spectra were being recorded at a variable delay between two sequential shots, following each other in pairs at a repetition rate of 10 Hz. The series of 500 coupled measurements, at the delays in the range 1 µs-10 ms and the displacements up to 2.5 mm, have been performed in a few points of an open premixed methane-air flame of a laboratory burner with the time-averaged temperatures in the range 1200-1800 K. From the spectra, "instantaneous" temperatures, at the given delay and probe volume distance, have been derived. This allowed the auto-correlation coefficients of temperature fluctuations versus the delay and the displacement to be calculated. These dependences enabled to evaluate temperature correlation times and lengths under various mixture flow rates and equivalence ratios.

Microscopic Cascading Devices for Boosting Mucus Penetration in Oral Drug Delivery-Micromotors Nesting Inside Microcontainers.

In the case of macromolecules and poorly permeable drugs, oral drug delivery features low bioavailability and low absorption across the intestinal wall. Intestinal absorption can be improved if the drug formulation could be transported close to the epithelium. To achieve this, a cascade delivery device comprising Magnesium-based Janus micromotors (MMs) nesting inside a microscale containers (MCs) has been conceptualized. The device aims at facilitating targeted drug delivery mediated by MMs that can lodge inside the intestinal mucosa. Loading MMs into MCs can potentially enhance drug absorption through increased proximity and unidirectional release. The MMs will be provided with optimal conditions for ejection into any residual mucus layer that the MCs have not penetrated. MMS confined inside MCs propel faster in the mucus environment as compared to non-confined MMs. Upon contact with a suitable fuel, the MM-loaded MC itself can also move. An in vitro study shows fast release profiles and linear motion properties in porcine intestinal mucus compared to more complex motion in aqueous media. The concept of dual-acting cascade devices holds great potential in applications where proximity to epithelium and deep mucus penetration are needed.

Dissolvable microneedles for transdermal drug delivery showing skin pentation and modified drug release.

Topical therapies for chronic skin diseases suffer from a low patient compliance due to the inconvenient treatment regimens of available products. Dissolvable microneedles (MN) with modified release offer an interesting possibility to increase the compliance by acting as a depot in the skin and thereby decreasing the dosing frequency. Furthermore, the bioavailability can be increased significantly by bypassing the barrier of the skin by the direct penetration of the MN into the skin. In this study the depot effect and skin penetration of an innovative dissolvable MN patch was assessed by insertion in ex vivo human skin and in vivo using minipigs. The MN patches are based on biodegradable polymers and the active pharmaceutical ingredients calcipotriol (Calci) and betamethasone-17-21-dipropionate (BDP) used to treat psoriasis. Using computed tomography (CT) and Coherent anti-Stokes Raman scattering (CARS) microscopy it was possible to visualize the skin penetration and follow the morphology of the MN as function of time in the skin. The depot effect was assessed by studying the modified in vitro release in an aqueous buffer and by comparing the drug release of a single application of a patch both ex vivo and in vivo to daily application of a marketed oleogel containing the same active pharmaceutical ingredients. The CT and CARS images showed efficient penetration of the MN patches into the upper dermis and a slow swelling process of the drug containing tip over a period of 8 days. Furthermore, CARS demonstrated that it can be used as a noninvasive technique with potential applicability in clinical settings. The in vitro release studies show a release of 54% over a time period of 30 days. The pharmacological relevance of MNs was confirmed in human skin explants and in vivo after single application and showed a similar response on calcipotriol and BDP mediated signaling events compared to daily application of the active oleogel. Altogether it was demonstrated that the MN can penetrate the skin and have the potential to provide a depot effect.

Comparative analysis of dermal collagen and lipids in cereblon ablated mice using a multimodal nonlinear optical system.

By utilizing a multimodal nonlinear optical system that combines coherent anti-Stokes Raman scattering and second harmonic generation to investigate biological characteristics of dermal tissues ex vivo, we demonstrate the potential feasibility of using this optical approach as a powerful new investigative tool for future biomedical research. For this study, our optical system was utilized for the first time to analyze lipid and collagen profiles in cereblon knockout (KO) mouse skin, and we were able to discover significant alterations in the number of carbon-carbon double bonds (wild-type vs. cereblon KO; NCC : 0.75 vs. 0.85) of skin fatty acids in triacylglycerides as well as changes in dermal collagen fibers (25% reduction in cereblon KO). By adopting our optical system to biological studies, we provide researchers with another diagnostic approach to validate their experimental results, which will significantly advance the state of biomedical research.

Bessel Beam Coherent Anti-Stokes Raman Scattering Spectroscopy for Turbulent Flow Diagnosis.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy plays an important role in chemical analysis for transient flow dynamics. Due to the turbulent ambient conditions, the CARS spectrum often suffers from a poor signal-to-noise ratio (SNR) and cannot provide a convincing measurement. Here, we report on a CARS spectroscopic method using a Bessel beam to enhance the spectral fidelity and SNR in a quasi-turbulent environment. Compared with traditional CARS, the measurement accuracy is significantly improved by taking advantage of the anti-scattering and self-healing characteristics of the Bessel beam. Our preliminary results indicate that Bessel beam CARS could be a promising method for high precision turbulent flow measurement fields.

Development of an image classification pipeline for atherosclerotic plaques assessment using supervised machine learning.

BACKGROUND: During atherosclerosis, the narrowing of the arterial lumen is observed through the accumulation of bio compounds and the formation of plaque within artery walls. A non-linear optical imaging modality (NLOM), coherent anti-stokes Raman scattering (CARS) microscopy, can be used to image lipid-rich structures commonly found in atherosclerotic plaques. By matching the lipid's molecular vibrational frequencies (CH bonds), it is possible to map the accumulation of lipid-rich structures without the need for exogenous labelling and/or processing of the samples. CARS allows for the visualization of the morphological features of plaque. In combination with supervised machine learning, CARS imaged morphological features can be used to characterize the progression of atherosclerotic plaques.  RESULTS: Based on a set of label-free CARS images of atherosclerotic plaques (i.e. foam cell clusters) from a Watanabe heritable hyperlipidemic rabbit model, we developed an automated pipeline to classify atherosclerotic lesions based on their major morphological features. Our method uses image preprocessing to first improve the quality of the CARS-imaged plaque, followed by the segmentation of the plaque using Otsu thresholding, marker-controlled watershed, K-means segmentation and a novel independent foam cell thresholding segmentation. To define relevant morphological features, 27 quantitative features were extracted and further refined by a novel coefficient of variation feature refinement method in accordance with filter-type feature selection. Refined morphological features were supplied into three supervised machine learning algorithms; K-nearest neighbour, support vector machine and decision tree classifier. The classification pipeline showcased the ability to exploit relevant plaque morphological features to accurately classify 3 pre-defined stages of atherosclerosis: early fatty streak development (EFS) and advancing atheroma (AA) with a greater than 85% class accuracy CONCLUSIONS: Through the combination of CARS microscopy and computational methods, a powerful classification tool was developed to identify the progression of atherosclerotic plaque in an automated manner. Using a curated dataset, the classification pipeline demonstrated the ability to differentiate between EFS, EF and AA. Thus, presenting the opportunity to classify the onset of atherosclerosis at an earlier stage of development.

Long-Term Repeatable In Vivo Monitoring of Amyloid-ß Plaques and Vessels in Alzheimer's Disease Mouse Model with Combined TPEF/CARS Microscopy.

Long-term, repeatable monitoring of the appearance and progress of Alzheimer's disease (AD) in real time can be extremely beneficial to acquire highly reliable diagnostic insights, which is crucial for devising apt strategies towards effective AD treatment. Herein, we present an optimized innovative cranial window imaging method for the long-term repeatable imaging of amyloid-ß (Aß) plaques and vessels in an AD mouse model. Basically, two-photon excitation fluorescence (TPEF) microscopy was used to monitor the fluorescently labeled Aß plaques, whereas the label-free blood vessels were studied using coherent anti-Stokes Raman scattering (CARS) microscopy in the live in vivo AD mouse model. It was possible to clearly observe the Aß deposition and vascular structure in the target cortex localization for 31 weeks in the AD mouse model using this method. The combined TPEF/CARS imaging studies were also instrumental in realizing the relationship between the tendency of Aß deposition and ageing. Essentially, the progression of cerebral amyloid angiopathy (CAA) in the AD mouse model was quantitatively characterized, which revealed that the proportion Aß deposition in the unit vessel can increase from 13.63% to 28.80% upon increasing the age of mice from 8 months old to 14 months old. The proposed imaging method provided an efficient, safe, repeatable platform with simple target localization aptitude towards monitoring the brain tissues, which is an integral part of studying any brain-related physiological or disease conditions to extract crucial structural and functional information.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Enhancement of Molecular Coherent Anti-Stokes Raman Scattering with Silicon Nanoantennas.

Surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS) takes advantage of surface plasmon resonances supported on metallic nanostructures to amplify the coherent Raman response of target molecules. While these metallic antennas have found significant success in SE-CARS studies, photoinduced morphological changes to the nanoantenna under ultrafast excitation introduce significant hurdles in terms of stability and reproducilibty. These hurdles need to be overcome in order to establish SE-CARS as a reliable tool for rapid biomolecular sensing. Here, we address this challenge by performing molecular CARS measurements enhanced by nanoantennas made from high-index dielectric particles with more favorable thermal properties. We present the first experimental demonstration of enhanced molecular CARS signals observed at Si nanoantennas, which offer much improved thermal stability compared to their metallic counterparts.

Changes in hydration of liposome membranes exposed to nanosecond electric pulses detected by wide-field Coherent anti-Stokes Raman microspectroscopy.

Electropulsation has become a powerful technological platform for electromanipulation of cells and tissues for various medical and biotechnological applications, but the molecular changes that underlay the very first initiation step of this process have not been experimentally observed. Here, we endowed a wide-field Coherent anti-Stokes Raman Scattering platform with an ad-hoc electromagnetic exposure device and we demonstrated, using artificial lipid vesicles (i.e. liposomes), that electropulsation is initiated by the increase of interstitial water content in liposome membranes. A pulse-dependent accumulation of the interstitial water molecules is observed in the membranes and a plausible mechanism supported by a computational electrochemical model is presented and discussed.

CARS Imaging Advances Early Diagnosis of Cardiac Manifestation of Fabry Disease.

Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient's prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90-96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.

Imaging approaches for monitoring three-dimensional cell and tissue culture systems.

The past decade has seen an increasing demand for more complex, reproducible and physiologically relevant tissue cultures that can mimic the structural and biological features of living tissues. Monitoring the viability, development and responses of such tissues in real-time are challenging due to the complexities of cell culture physical characteristics and the environments in which these cultures need to be maintained in. Significant developments in optics, such as optical manipulation, improved detection and data analysis, have made optical imaging a preferred choice for many three-dimensional (3D) cell culture monitoring applications. The aim of this review is to discuss the challenges associated with imaging and monitoring 3D tissues and cell culture, and highlight topical label-free imaging tools that enable bioengineers and biophysicists to non-invasively characterise engineered living tissues.

Polystyrene microbeads influence lipid storage distribution in C. elegans as revealed by coherent anti-Stokes Raman scattering (CARS) microscopy.

The exposure of Caenorhabditis elegans to polystyrene (PS) beads of a wide range of sizes impedes feeding, by reducing food consumption, and has been linked to inhibitory effects on the reproductive capacity of this nematode, as determined in standardized toxicity tests. Lipid storage provides energy for longevity, growth, and reproduction and may influence the organismal response to stress, including the food deprivation resulting from microplastics exposure. However, the effects of microplastics on energy storage have not been investigated in detail. In this study, C. elegans was exposed to ingestible sizes of PS beads in a standardized toxicity test (96 h) and in a multigeneration test (∼21 days), after which lipid storage was quantitatively analyzed in individual adults using coherent anti-Stokes Raman scattering (CARS) microscopy. The results showed that lipid storage distribution in C. elegans was altered when worms were exposed to microplastics in form of PS beads. For example, when exposed to 0.1-µm PS beads, the lipid droplet count was 93% higher, the droplets were up to 56% larger, and the area of the nematode body covered by lipids was up to 79% higher than in unexposed nematodes. The measured values tended to increase as PS bead sizes decreased. Cultivating the nematodes for 96 h under restricted food conditions in the absence of beads reproduced the altered lipid storage and suggested that it was triggered by food deprivation, including that induced by the dilutional effects of PS bead exposure. Our study demonstrates the utility of CARS microscopy to comprehensively image the smaller microplastics (<10 µm) ingested by nematodes and possibly other biota in investigations of the effects at the level of the individual organism.