Imaging Metabolic Flow of Water in Plants with Isotope-Traced Stimulated Raman Scattering Microscopy.

Water plays a vital role in the life cycle of plants, participating in various critical biochemical reactions during both non-photosynthetic and photosynthetic processes. Direct visualization of the metabolic activities of water in plants with high spatiotemporal resolution is essential to reveal the functional utilization of water. Here, stimulated Raman scattering (SRS) microscopy is applied to monitor the metabolic processes of deuterated water (D2O) in model plant Arabidopsis thaliana (A. thaliana). The work shows that in plants uptaking D2O/water solution, proton-transfer from water to organic metabolites results in the formation of C-D bonds in newly synthesized biomolecules (lipid, protein, and polysaccharides, etc.) that allow high-resolution detection with SRS. Reversible metabolic pathways of oil-starch conversion between seed germination and seed development processes are verified. Spatial heterogeneity of metabolic activities along the vertical axis of plants (root, stem, and tip meristem), as well as the radial distributions of secondary growth on the horizontal cross-sections are quantified. Furthermore, metabolic flow of protons from plants to animals is visualized in aphids feeding on A. thaliana. Collectively, SRS microscopy has potential to trace a broad range of matter flows in plants, such as carbon storage and nutrition metabolism.

Self-stacked small molecules for ultrasensitive, substrate-free Raman imaging in vivo.

Raman spectroscopy using surface-enhanced Raman scattering (SERS) nanoprobes represents an ultrasensitive and high-precision technique for in vivo imaging. Clinical translation of SERS nanoprobes has been hampered by biosafety concerns about the metal substrates used to enhance Raman signals. We report a set of small molecules with bis-thienyl-substituted benzobisthiadiazole structures that enhance Raman signal through self-stacking rather than external substrates. In our technique, called stacking-induced charge transfer-enhanced Raman scattering (SICTERS), the self-stacked small molecules form an ordered spatial arrangement that enables three-dimensional charge transfer between neighboring molecules. The Raman scattering cross-section of SICTERS nanoprobes is 1350 times higher than that of conventional SERS gold nanoprobes of similar particle size. SICTERS outperforms SERS in terms of in vivo imaging sensitivity, resolution and depth. SICTERS is capable of noninvasive Raman imaging of blood and lymphatic vasculatures, which has not been achieved by SERS. SICTERS represents an alternative technique to enhance Raman scattering for guiding the design of ultrasensitive substrate-free Raman imaging probes.

Nanoplastics Detected in Commercial Sea Salt.

People of all ages consume salt every day, but is it really just salt? Plastic nanoparticles [nanoplastics (NPs)] pose an increasing environmental threat and have begun to contaminate everyday salt in consumer goods. Herein, we developed a combined surface enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) approach that can realize the filtration, enrichment, and detection of NPs in commercial salt. The Au-loaded (50 nm) anodic alumina oxide substrate was used as the SERS substrate to explore the potential types of NP contaminants in salts. SRS was used to conduct imaging and quantify the presence of the NPs. SRS detection was successfully established through standard plastics, and NPs were identified through the match of the hydrocarbon group of the nanoparticles. Simultaneously, the NPs were quantified based on the high spatial resolution and rapid imaging of the SRS imaging platform. NPs in sea salts produced in Asia, Australasia, Europe, and the Atlantic were studied. We estimate that, depending on the location, an average person could be ingesting as many as 6 million NPs per year through the consumption of sea salt alone. The potential health hazards associated with NP ingestion should not be underestimated.

Spatiotemporal Observation of Monosodium Urate Crystals Deposition in Synovial Organoids Using Label-Free Stimulated Raman Scattering.

Gout, a common form of arthritis, is characterized by the deposition of monosodium urate (MSU) crystals in joints. MSU deposition in synovial tissues would initiate arthritis flares and recurrence, causing irreversible joint damage. However, the dynamic deposition of MSU crystals in tissues lacks experimental observation. In this study, we used chemical-specific, label-free stimulated Raman scattering (SRS) microscopy to investigate the spatiotemporal deposition and morphological characteristics of MSU crystals in human synovial organoids. Our findings revealed a critical 12-h window for MSU deposition in the lining layer of gouty synovium. Moreover, distinctive inflammatory reactions of the lining and sublining synovial layers in gout using SRS microscopy were further verified by immunofluorescence. Importantly, we identified a crucial proinflammatory role of sublining fibroblast-like synoviocytes, indicating a need for targeted medication treatment on these cells. Our work contributes to the fundamental understanding of MSU-based diseases and offers valuable insights for the future development of targeted gout therapies.

TET2-STAT3-CXCL5 nexus promotes neutrophil lipid transfer to fuel lung adeno-to-squamous transition.

Phenotypic plasticity is a rising cancer hallmark, and lung adeno-to-squamous transition (AST) triggered by LKB1 inactivation is significantly associated with drug resistance. Mechanistic insights into AST are urgently needed to identify therapeutic vulnerability in LKB1-deficient lung cancer. Here, we find that ten-eleven translocation (TET)-mediated DNA demethylation is elevated during AST in KrasLSL-G12D/+; Lkb1L/L (KL) mice, and knockout of individual Tet genes reveals that Tet2 is required for squamous transition. TET2 promotes neutrophil infiltration through STAT3-mediated CXCL5 expression. Targeting the STAT3-CXCL5 nexus effectively inhibits squamous transition through reducing neutrophil infiltration. Interestingly, tumor-infiltrating neutrophils are laden with triglycerides and can transfer the lipid to tumor cells to promote cell proliferation and squamous transition. Pharmacological inhibition of macropinocytosis dramatically inhibits neutrophil-to-cancer cell lipid transfer and blocks squamous transition. These data uncover an epigenetic mechanism orchestrating phenotypic plasticity through regulating immune microenvironment and metabolic communication, and identify therapeutic strategies to inhibit AST.

Virtual formalin-fixed and paraffin-embedded staining of fresh brain tissue via stimulated Raman CycleGAN model.

Intraoperative histology is essential for surgical guidance and decision-making. However, frozen-sectioned hematoxylin and eosin (H&E) staining suffers from degraded accuracy, whereas the gold-standard formalin-fixed and paraffin-embedded (FFPE) H&E is too lengthy for intraoperative use. Stimulated Raman scattering (SRS) microscopy has shown rapid histology of brain tissue with lipid/protein contrast but is challenging to yield images identical to nucleic acid-/protein-based FFPE stains interpretable to pathologists. Here, we report the development of a semi-supervised stimulated Raman CycleGAN model to convert fresh-tissue SRS images to H&E stains using unpaired training data. Within 3 minutes, stimulated Raman virtual histology (SRVH) results that matched perfectly with true H&E could be generated. A blind validation indicated that board-certified neuropathologists are able to differentiate histologic subtypes of human glioma on SRVH but hardly on conventional SRS images. SRVH may provide intraoperative diagnosis superior to frozen H&E in both speed and accuracy, extendable to other types of solid tumors.

Imaging Ultrafast Dynamics of Pressure-Driven Phase Transitions in Black Phosphorus and Anomalous Coherent Phonon Softening.

Applying high pressure to effectively modulate the electronic and lattice structures of materials could unravel various physical properties associated with phase transitions. In this work, high-pressure-compatible femtosecond pump-probe microscopy was constructed to study the pressure-dependent ultrafast dynamics in black phosphorus (BP) thin films. We observed pressure-driven evolution of the electronic topological transition and three structural phases as the pressure reached ∼22 GPa, which could be clearly differentiated in the transient absorption images containing spatially resolved ultrafast carrier and coherent phonon dynamics. Surprisingly, an anomalous coherent acoustic phonon mode with pressure softening behavior was observed within the range of ∼3-8 GPa, showing distinct laser power and time dependences. Density functional theory calculations show that this mode, identified as the shear mode along the armchair orientation, gains significant electron-phonon coupling strength from out-of-plane compression that leads to decreased phonon frequency. Our results provide insights into the structure evolution of BP with pressure and hold potential for applications in microelectromechanical devices.

Significantly Accelerated Photochemical Perfluorooctanoic Acid Decomposition at the Air-Water Interface of Microdroplets.

The efficient elimination of per- and polyfluoroalkyl substances (PFASs) from the environment remains a huge challenge and requires advanced technologies. Herein, we demonstrate that perfluorooctanoic acid (PFOA) photochemical decomposition could be significantly accelerated by simply carrying out this process in microdroplets. The almost complete removal of 100 and 500 µg/L PFOA was observed after 20 min of irradiation in microdroplets, while this was achieved after about 2 h in the corresponding bulk phase counterpart. To better compare the defluorination ratio, 10 mg/L PFOA was used typically, and the defluorination rates in microdroplets were tens of times faster than that in the bulk phase reaction system. The high performances in actual water matrices, universality, and scale-up applicability were demonstrated as well. We revealed in-depth that the great acceleration is due to the abundance of the air-water interface in microdroplets, where the reactants concentration enrichment, ultrahigh interfacial electric field, and partial solvation effects synergistically promoted photoreactions responsible for PFOA decomposition, as evidenced by simulated Raman scattering microscopy imaging, vibrational Stark effect measurement, and DFT calculation. This study provides an effective approach and highlights the important roles of air-water interface of microdroplets in PFASs treatment.

Nondestructive Nonlinear Optical Microscopy Revealed the Blackening Mechanism of Ancient Chinese Jades.

Jade is most valued in Chinese culture since ancient times. For unearthed jade artifacts, the alteration color resulting from weathering effects and human activities provides information for cultural heritage conservation, archaeology, and history. Currently, the noninvasive 3-dimensional characterization of jade artifacts with high chemical and spatial resolution remains challenging. In this work, we applied femtosecond pump-probe microscopy and second harmonic generation microscopy techniques to study the black alteration of an ancient jade artifact of the late Spring and Autumn period (546 to 476 BC). The direct cause of the "mercury alteration" phenomena was discovered to be the conversion of metacinnabar from buried cinnabar in the tomb. Furthermore, a 3-dimensional optical reconstruction of the black alteration was achieved, providing a high-resolution method for analyzing the blackening mechanism without the need of sample damage. Our approach opens up new opportunities to extract microscopic spatiochemical information for a broad range of alteration colors in jade artifacts.

12-inch growth of uniform MoS2 monolayer for integrated circuit manufacture.

Two-dimensional (2D) semiconductors, such as transition metal dichalcogenides, provide an opportunity for beyond-silicon exploration. However, the lab to fab transition of 2D semiconductors is still in its preliminary stages, and it has been challenging to meet manufacturing standards of stability and repeatability. Thus, there is a natural eagerness to grow wafer-level, high-quality films with industrially acceptable scale-cost-performance metrics. Here we report an improved chemical vapour deposition synthesis method in which the controlled release of precursors and substrates predeposited with amorphous Al2O3 ensure the uniform synthesis of monolayer MoS2 as large as 12 inches while also enabling fast and non-toxic growth to reduce manufacturing costs. Transistor arrays were fabricated to further confirm the high quality of the film and its integrated circuit application potential. This work achieves the co-optimization of scale-cost-performance metrics and lays the foundation for advancing the integration of 2D semiconductors in industry-standard pilot lines.

Evaluating Differentiation Status of Mesenchymal Stem Cells by Label-Free Microscopy System and Machine Learning.

Mesenchymal stem cells (MSCs) play a crucial role in tissue engineering, as their differentiation status directly affects the quality of the final cultured tissue, which is critical to the success of transplantation therapy. Furthermore, the precise control of MSC differentiation is essential for stem cell therapy in clinical settings, as low-purity stem cells can lead to tumorigenic problems. Therefore, to address the heterogeneity of MSCs during their differentiation into adipogenic or osteogenic lineages, numerous label-free microscopic images were acquired using fluorescence lifetime imaging microscopy (FLIM) and stimulated Raman scattering (SRS), and an automated evaluation model for the differentiation status of MSCs was built based on the K-means machine learning algorithm. The model is capable of highly sensitive analysis of individual cell differentiation status, so it has great potential for stem cell differentiation research.

Imaging of pH distribution inside individual microdroplet by stimulated Raman microscopy.

Aerosol microdroplets as microreactors for many important atmospheric reactions are ubiquitous in the atmosphere. pH largely regulates the chemical processes within them; however, how pH and chemical species spatially distribute within an atmospheric microdroplet is still under intense debate. The challenge is to measure pH distribution within a tiny volume without affecting the chemical species distribution. We demonstrate a method based on stimulated Raman scattering microscopy to visualize the three-dimensional pH distribution inside single microdroplets of varying sizes. We find that the surface of all microdroplets is more acidic, and a monotonic trend of pH decreasing is observed in the 2.9-µm aerosol microdroplet from center to edge, which is well supported by molecular dynamics simulation. However, bigger cloud microdroplet differs from small aerosol for pH distribution. This size-dependent pH distribution in microdroplets can be related to the surface-to-volume ratio. This work presents noncontact measurement and chemical imaging of pH distribution in microdroplets, filling the gap in our understanding of spatial pH in atmospheric aerosol.

Imaging Low-Temperature Phases of Ice with Polarization-Resolved Hyperspectral Stimulated Raman Scattering Microscopy.

Water freezes into various phases of ice under different cryogenic temperatures and pressure conditions, such as ice Ih and ice XI at normal pressure. Vibrational imaging with high spectral, spatial, and polarization resolutions could provide detailed information on ice, including the phases and crystal orientations at the microscopic level. Here, we report in situ stimulated Raman scattering (SRS) imaging of ice to analyze the vibrational spectral changes of the OH stretching modes associated with the phase transition between ice Ih and ice XI. In addition, polarization-resolved measurements were performed to reveal the microcrystal orientations of the two phases of ice, with the spatial-dependent anisotropy pattern indicating the inhomogeneous distribution of their orientations. Furthermore, the angular patterns were theoretically explained by third-order nonlinear optics with the known crystal symmetries of the ice phases. Our work may provide new opportunities to investigate many intriguing physical chemistry properties of ice under low-temperature conditions.

Histological diagnosis of unprocessed breast core-needle biopsy via stimulated Raman scattering microscopy and multi-instance learning.

Core-needle biopsy (CNB) plays a vital role in the initial diagnosis of breast cancer. However, the complex tissue processing and global shortage of pathologists have hindered traditional histopathology from timely diagnosis on fresh biopsies. In this work, we developed a full digital platform by integrating label-free stimulated Raman scattering (SRS) microscopy with weakly-supervised learning for rapid and automated cancer diagnosis on un-labelled breast CNB. Methods: We first compared the results of SRS imaging with standard hematoxylin and eosin (H&E) staining on adjacent frozen tissue sections. Then fresh unprocessed biopsy tissues were imaged by SRS to reveal diagnostic histoarchitectures. Next, weakly-supervised learning, i.e., the multi-instance learning (MIL) model was conducted to evaluate the ability to differentiate between benign and malignant cases, and compared with the performance of supervised learning model. Finally, gradient-weighted class activation mapping (Grad-CAM) and semantic segmentation were performed to spatially resolve benign/malignant areas with high efficiency. Results: We verified the ability of SRS in revealing essential histological hallmarks of breast cancer in both thin frozen sections and fresh unprocessed biopsy, generating histoarchitectures well correlated with H&E staining. Moreover, we demonstrated that weakly-supervised MIL model could achieve superior classification performance to supervised learnings, reaching diagnostic accuracy of 95% on 61 biopsy specimens. Furthermore, Grad-CAM allowed the trained MIL model to visualize the histological heterogeneity within the CNB. Conclusion: Our results indicate that MIL-assisted SRS microscopy provides rapid and accurate diagnosis on histologically heterogeneous breast CNB, and could potentially help the subsequent management of patients.

Region-resolved multi-omics of the mouse eye.

The eye is a complex organ consisting of multiple compartments with unique and specialized properties, and small disturbances in one eye region can result in impaired vision and blindness. Although there have been advancements in ocular research, the hierarchical molecular network in region-wide resolution, indicating the division of labor and crosstalk among different eye regions, is not yet comprehensively illuminated. Here, we present an atlas of region-resolved proteome and lipidome of mouse eye. Multiphoton microscopy-guided laser microdissection combined with in-depth label-free proteomics identifies 13,536 proteins across various mouse eye regions. Further integrative analysis of spectral imaging, label-free proteome, and imaging mass spectrometry of the lipidome and phosphoproteome reveals distinctive molecular features, including proteins and lipids of various anatomical mouse eye regions. These deposited datasets and our open proteome server integrating all information provide a valuable resource for future functional and mechanistic studies of mouse eye and ocular disease.

Significantly Accelerated Hydroxyl Radical Generation by Fe(III)-Oxalate Photochemistry in Aerosol Droplets.

Fe(III)-oxalate complexes are ubiquitous in atmospheric environments, which can release reactive oxygen species (ROS) such as H2O2, O•2-, and OH• under light irradiation. Although Fe(III)-oxalate photochemistry has been investigated extensively, the understanding of its involvement in authentic atmospheric environments such as aerosol droplets is far from enough, since the current available knowledge has mainly been obtained in bulk-phase studies. Here, we find that the production of OH• by Fe(III)-oxalate in aerosol microdroplets is about 10-fold greater than that of its bulk-phase counterpart. In addition, in the presence of Fe(III)-oxalate complexes, the rate of photo-oxidation from SO2 to sulfate in microdroplets was about 19-fold faster than that in the bulk phase. The availability of efficient reactants and mass transfer due to droplet effects made dominant contributions to the accelerated OH• and SO42- formation. This work highlights the necessary consideration of droplet effects in atmospheric laboratory studies and model simulations.

Stimulated Raman Scattering Microscopy Enables Gleason Scoring of Prostate Core Needle Biopsy by a Convolutional Neural Network.

Focal therapy (FT) has been proposed as an approach to eradicate clinically significant prostate cancer while preserving the normal surrounding tissues to minimize treatment-related toxicity. Rapid histology of core needle biopsies is essential to ensure the precise FT for localized lesions and to determine tumor grades. However, it is difficult to achieve both high accuracy and speed with currently available histopathology methods. Here, we demonstrated that stimulated Raman scattering (SRS) microscopy could reveal the largely heterogeneous histologic features of fresh prostatic biopsy tissues in a label-free and near real-time manner. A diagnostic convolutional neural network (CNN) built based on images from 61 patients could classify Gleason patterns of prostate cancer with an accuracy of 85.7%. An additional 22 independent cases introduced as external test dataset validated the CNN performance with 84.4% accuracy. Gleason scores of core needle biopsies from 21 cases were calculated using the deep learning SRS system and showed a 71% diagnostic consistency with grading from three pathologists. This study demonstrates the potential of a deep learning-assisted SRS platform in evaluating the tumor grade of prostate cancer, which could help simplify the diagnostic workflow and provide timely histopathology compatible with FT treatment. SIGNIFICANCE: A platform combining stimulated Raman scattering microscopy and a convolutional neural network provides rapid histopathology and automated Gleason scoring on fresh prostate core needle biopsies without complex tissue processing.