Rapid single-particle chemical imaging of nanoplastics by SRS microscopy.

Plastics are now omnipresent in our daily lives. The existence of microplastics (1 µm to 5 mm in length) and possibly even nanoplastics (<1 µm) has recently raised health concerns. In particular, nanoplastics are believed to be more toxic since their smaller size renders them much more amenable, compared to microplastics, to enter the human body. However, detecting nanoplastics imposes tremendous analytical challenges on both the nano-level sensitivity and the plastic-identifying specificity, leading to a knowledge gap in this mysterious nanoworld surrounding us. To address these challenges, we developed a hyperspectral stimulated Raman scattering (SRS) imaging platform with an automated plastic identification algorithm that allows micro-nano plastic analysis at the single-particle level with high chemical specificity and throughput. We first validated the sensitivity enhancement of the narrow band of SRS to enable high-speed single nanoplastic detection below 100 nm. We then devised a data-driven spectral matching algorithm to address spectral identification challenges imposed by sensitive narrow-band hyperspectral imaging and achieve robust determination of common plastic polymers. With the established technique, we studied the micro-nano plastics from bottled water as a model system. We successfully detected and identified nanoplastics from major plastic types. Micro-nano plastics concentrations were estimated to be about 2.4 ± 1.3 × 105 particles per liter of bottled water, about 90% of which are nanoplastics. This is orders of magnitude more than the microplastic abundance reported previously in bottled water. High-throughput single-particle counting revealed extraordinary particle heterogeneity and nonorthogonality between plastic composition and morphologies; the resulting multidimensional profiling sheds light on the science of nanoplastics.

Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy.

Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.

Quantitative Label-Free Chemical Imaging of PLGA Nanoparticles in Cells and Tissues with Single-Particle Sensitivity.

Nanomedicine has brought significant advancements to healthcare by utilizing nanotechnology in medicine. Despite much promise, the further development of nanocarriers for clinical use has been hindered by a lack of understanding and visualization of nano-bio interactions. Conventional imaging methods have limitations in resolution, sensitivity, and specificity. This study introduces a label-free optical approach using stimulated Raman scattering (SRS) microscopy to image poly(lactic-co-glycolic acid) (PLGA) nanocarriers, the most widely used polymeric nanocarrier for delivery therapeutic agents, with single-particle sensitivity and quantification capabilities. A unique Raman peak was identified for PLGA ester, enabling generalized bio-orthogonal bond imaging. We demonstrated quantitative SRS imaging of PLGA nanocarriers across different biological systems from cells to animal tissues. This label-free imaging method provides a powerful tool for studying this prevalent nanocarrier and quantitatively visualizing their distribution, interaction, and clearance in vivo.

Cyano-Hydrol green derivatives: Expanding the 9-cyanopyronin-based resonance Raman vibrational palette.

9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.

Stimulated Raman Scattering Microscopy Reveals Bioaccumulation of Small Microplastics in Protozoa from Natural Waters.

Microplastics (MPs) are pollutants of global concern, and bioaccumulation determines their biological effects. Although microorganisms form a large fraction of our ecosystem's biomass and are important in biogeochemical cycling, their accumulation of MPs has never been confirmed in natural waters because current tools for field biological samples can detect only MPs > 10 µm. Here, we show that stimulated Raman scattering microscopy (SRS) can image and quantify the bioaccumulation of small MPs (<10 µm) in protozoa. Our label-free method, which differentiates MPs by their SRS spectra, detects individual and mixtures of different MPs (e.g., polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, and poly(methyl methacrylate)) in protozoa. The ability of SRS to quantify cellular MP accumulation is similar to that of flow cytometry, a fluorescence-based method commonly used to determine cellular MP accumulation. Moreover, we discovered that protozoa in water samples from Yangtze River, Xianlin Wastewater Treatment Plant, Lake Taihu and the Pearl River Estuary accumulated MPs < 10 µm, but the proportion of MP-containing cells was low (∼2-5%). Our findings suggest that small MPs could potentially enter the food chain and transfer to organisms at higher trophic levels, posing environmental and health risks that deserve closer scrutiny.

Characterization of polyphosphate dynamics in the widespread freshwater diatom Achnanthidium minutissimum under varying phosphorus supplies.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell-1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (Pi). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P(+)), or P deplete (P(-)) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after Pi resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom.

Single-mode characteristic of a supermode microcavity Raman laser.

Microlasers in near-degenerate supermodes lay the cornerstone for studies of non-Hermitian physics, novel light sources, and advanced sensors. Recent experiments of the stimulated scattering in supermode microcavities reported beating phenomena, interpreted as dual-mode lasing, which, however, contradicts their single-mode nature due to the clamped pump field. Here, we investigate the supermode Raman laser in a whispering-gallery microcavity and demonstrate experimentally its single-mode lasing behavior with a side-mode suppression ratio (SMSR) up to 37 dB, despite the emergence of near-degenerate supermodes by the backscattering between counterpropagating waves. Moreover, the beating signal is recognized as the transient interference during the switching process between the two supermode lasers. Self-injection is exploited to manipulate the lasing supermodes, where the SMSR is further improved by 15 dB and the laser linewidth is below 100 Hz.

Hydrated Electron Dynamics and Stimulated Raman Scattering in Water Induced by Ultrashort Laser Pulses.

For this study, we employed intense 400 nm, 100 fs pulses linearly propagated through a 50 cm water medium, initially self-stretching the excitation pulses to 2.50 ps. Subsequently, the self-stretched 2.50 ps pulses were focused into deionized water, and we conducted transient absorption experiments to measure and investigate the dynamics of hydrated electrons in water. The excess electrons generated were injected into the hydrogen bond network of the water cluster, leading to the observation of saturated hydrated electrons. Additionally, we observed the emergence of the forward stimulated Raman scattering (SRS) of water molecules. We report the experimental observation of a weak forward SRS emission at 463 nm (corresponding to 3400 cm-1), indicative of the ordinary OH stretching vibration in the liquid phase. Moreover, we observed an intense forward SRS emission at 460 nm in water, corresponding to two anomalous Raman shifts at 3260 cm-1 and 3355 cm-1. These anomalous Raman shifts resulted from changes in the hydrogen bond network structure. We determine that the formation of not fully hydrated and saturated hydrated electrons plays a crucial role in producing this phenomenon.

Multiscale microscopy to decipher plant cell structure and dynamics.

New imaging methodologies with high contrast and molecular specificity allow researchers to analyze dynamic processes in plant cells at multiple scales, from single protein and RNA molecules to organelles and cells, to whole organs and tissues. These techniques produce informative images and quantitative data on molecular dynamics to address questions that cannot be answered by conventional biochemical assays. Here, we review selected microscopy techniques, focusing on their basic principles and applications in plant science, discussing the pros and cons of each technique, and introducing methods for quantitative analysis. This review thus provides guidance for plant scientists in selecting the most appropriate techniques to decipher structures and dynamic processes at different levels, from protein dynamics to morphogenesis.

Probe Beam Dichroism and Birefringence in Stimulated Raman Scattering in Polyatomic Molecules.

Dichroism and birefringence in Stimulated Raman Scattering (SRS) in polyatomic molecules were studied theoretically. General expressions describing the change of the polarization matrix of the probe laser beam transmitted through initially isotropic molecular sample excited by the pump laser beam have been derived. Arbitrary polarization states and propagation directions of the incoming pump and probe beams were considered. The expressions were written in terms of spherical tensor operators that allowed for separation of the field polarization tensor and the molecular part containing three scalar values of nonlinear optical susceptibility χ K p u 3 ${{\chi }_{{K}_{pu}}^{\left(3\right)}}$ with K p u ${{K}_{pu}}$ =0,1,2. The geometry of almost collinear propagation of the pump and probe beams through the molecular sample was considered in greater details. It was shown that the dichroism and birefringence refer to the nonlinear optical susceptibility element χ 2 3 ${{\chi }_{2}^{\left(3\right)}}$ and that their contributions to the SRS signal can be separated experimentally by using an appropriate probe beam polarization analyzer installed in front of the photodetector. Particular cases of the off-resonant SRS and resonant SRS have been considered. The results obtained were expressed in terms of the Stokes polarization parameters of the pump and probe beams.

Characterizing Silicone Oil-Induced Protein Aggregation with Stimulated Raman Scattering Imaging.

Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Identification and quantification of particles in drug products are important to understand particle formation mechanisms, which can help develop control strategies for particle formation during the formulation development and manufacturing process. However, existing analytical techniques such as microflow imaging and light obscuration measurement lack the sensitivity and resolution to detect particles with sizes smaller than 2 µm. More importantly, these techniques are not able to provide chemical information to determine particle composition. In this work, we overcome these challenges by applying the stimulated Raman scattering (SRS) microscopy technique to monitor the C-H Raman stretching modes of the proteinaceous particles and silicone oil droplets formed in the prefilled syringe barrel. By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein-silicone oil aggregates. We further show that morphological features are poor indicators of particle composition. Our method has the capability to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.

A novel chalcone derivative exerts anticancer effects by promoting apoptotic cell death of human pancreatic cancer cells.

Aggressive pancreatic cancer is typically treated using chemotherapeutics to reduce the tumor pre-operatively and prevent metastasis post-operatively, as well as surgical approaches. In the present study, we synthesized a hydroxyl group-introduced chalcone derivative (1, IC50 = 32.1 µM) and investigated its potential as an anticancer drug candidate by evaluating its apoptosis-promoting effects on BXPC-3 cancer cells. The viability of BXPC-3 cells treated with 1 was measured using the water-soluble tetrazolium 1 reagent. BXPC-3 cells induced by 1 were stained with diverse probes or antibodies, such as ethidium homodimer-1, Hoechst, anti-Ki67, and MitoTracker. Protein expression was measured using an immunoblotting assay, and mRNA expression was determined using real-time polymerase chain reaction. Apoptotic molecular features, such as lipid accumulation and protein degradation, were monitored directly using stimulated Raman scattering microspectroscopy. Through incubation time- and concentration-dependent studies of 1, we found that it significantly reduced the proliferation and increased the number of apoptotic BXPC-3 cells. Compound 1 induced mitochondrial dysfunction, phosphorylation of p38, and caspase 3 cleavage. These results indicate that 1 is a potential therapeutic agent for pancreatic cancer, providing valuable insights into the development of new anticancer drug candidates.

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy.

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Continuous-Wave Raman Lasing from Metal-Linked Organic Dimer Microcrystals.

Stimulated Raman scattering offers an alternative strategy to explore continuous-wave (c.w.) organic lasers, which, however, still suffers from the limitation of inadequate Raman gain in organic material systems. Here we propose a metal-linking approach to enhance the Raman gain of organic molecules. Self-assembled microcrystals of the metal linked organic dimers exhibit large Raman gain, therefore allowing for c.w. Raman lasing. Furthermore, broadband tunable Raman lasing is achieved in the organic dimer microcrystals by adjusting excitation wavelengths. This work advances the understanding of Raman gain in organic molecules, paving a way for the design of c.w. organic lasers.

Time-course quantitative mapping of caffeine within the epidermis, using high-contrast pump-probe stimulated Raman scattering microscopy.

BACKGROUND: An assessment of the drug penetration and distribution profiles within the skin is essential in dermatology and cosmetology. Recent advances in label-free imaging technologies have facilitated the direct detection of unlabeled compounds in tissues, with high resolution. However, it remains challenging to provide quantitative time-course distribution maps of drugs within the complex skin tissue. The present study aims at acquiring the real-time quantitative skin penetration profiles of topically applied caffeine, by means of a combination of pump-probe phase-modulated stimulated Raman scattering (PM-SRS) and confocal reflection microscopy. The recently developed PM-SRS microscopy is a unique imaging tool that can minimize strong background signals through a pulse-shaping technique, while providing high-contrast images of small molecules in tissues. MATERIALS AND METHODS: Reconstructed human skin epidermis models were used in order to analyze caffeine penetration in tissues. The penetration profiles of caffeine in an aqueous solution, an oil-in-water gel, and a water-in-oil gel were examined by combining PM-SRS and confocal reflection microscopy. RESULTS: The characteristic Raman signal of caffeine was directly detected in the skin model using PM-SRS. Integrating PM-SRS and confocal reflection microscopy allowed real-time concentration maps of caffeine to be obtained from formulation samples, within the skin model. Compared with the conventional Raman detection method, PM-SRS lowered the background tissue-oriented signals and supplied high-contrast images of caffeine. CONCLUSION: We successfully established real-time skin penetration profiles of caffeine from different formulations. PM-SRS microscopy proved to be a powerful, non-invasive, and real-time depth-profile imaging technique for use in quantitative studies of topically applied drugs.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Properties and Applications of Random Lasers as Emerging Light Sources and Optical Sensors: A Review.

In a random laser (RL), optical feedback arises from multiple scattering instead of conventional mirrors. RLs generate a laser-like emission, and meanwhile take advantage of a simpler and more flexible laser configuration. The applicability of RLs as light sources and optical sensors has been proved. These applications have been extended to the biological field, with tissues as natural scattering materials. Herein, the current state of the RL properties and applications was reviewed.

Label-free brain tumor imaging using Raman-based methods.

INTRODUCTION: Label-free Raman-based imaging techniques create the possibility of bringing chemical and histologic data into the operation room. Relying on the intrinsic biochemical properties of tissues to generate image contrast and optical tissue sectioning, Raman-based imaging methods can be used to detect microscopic tumor infiltration and diagnose brain tumor subtypes. METHODS: Here, we review the application of three Raman-based imaging methods to neurosurgical oncology: Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, and stimulated Raman histology (SRH). RESULTS: Raman spectroscopy allows for chemical characterization of tissue and can differentiate normal and tumor-infiltrated tissue based on variations in macromolecule content, both ex vivo and in vivo. To improve signal-to-noise ratio compared to conventional Raman spectroscopy, a second pulsed excitation laser can be used to coherently drive the vibrational frequency of specific Raman active chemical bonds (i.e. symmetric stretching of -CH2 bonds). Coherent Raman imaging, including CARS and stimulated Raman scattering microscopy, has been shown to detect microscopic brain tumor infiltration in fresh brain tumor specimens with submicron image resolution. Advances in fiber-laser technology have allowed for the development of intraoperative SRH as well as artificial intelligence algorithms to facilitate interpretation of SRH images. With molecular diagnostics becoming an essential part of brain tumor classification, preliminary studies have demonstrated that Raman-based methods can be used to diagnose glioma molecular classes intraoperatively. CONCLUSIONS: These results demonstrate how label-free Raman-based imaging methods can be used to improve the management of brain tumor patients by detecting tumor infiltration, guiding tumor biopsy/resection, and providing images for histopathologic and molecular diagnosis.

Stimulated Raman Scattering from Mie-Resonant Subwavelength Nanoparticles.

Resonant dielectric structures have emerged recently as a new platform for subwavelength nonplasmonic photonics. It was suggested and demonstrated that magnetic and electric Mie resonances can enhance substantially many effects at the nanoscale including spontaneous Raman scattering. Here, we demonstrate stimulated Raman scattering (SRS) for isolated crystalline silicon (c-Si) nanoparticles and observe experimentally a transition from spontaneous to stimulated scattering manifested in a nonlinear growth of the signal intensity above a certain pump threshold. At the Mie resonance, the light gets confined into a low volume of the resonant mode with enhanced electromagnetic fields inside the c-Si nanoparticle due to its high refractive index, which leads to an overall strong SRS signal at low pump intensities. Our finding paves the way for the development of efficient Raman nanolasers for multifunctional photonic metadevices.