Transient stimulated Raman scattering spectroscopy and imaging.

Stimulated Raman scattering (SRS) has been developed as an essential quantitative contrast for chemical imaging in recent years. However, while spectral lines near the natural linewidth limit can be routinely achieved by state-of-the-art spontaneous Raman microscopes, spectral broadening is inevitable for current mainstream SRS imaging methods. This is because those SRS signals are all measured in the frequency domain. There is a compromise between sensitivity and spectral resolution: as the nonlinear process benefits from pulsed excitations, the fundamental time-energy uncertainty limits the spectral resolution. Besides, the spectral range and acquisition speed are mutually restricted. Here we report transient stimulated Raman scattering (T-SRS), an alternative time-domain strategy that bypasses all these fundamental conjugations. T-SRS is achieved by quantum coherence manipulation: we encode the vibrational oscillations in the stimulated Raman loss (SRL) signal by femtosecond pulse-pair sequence excited vibrational wave packet interference. The Raman spectrum was then achieved by Fourier transform of the time-domain SRL signal. Since all Raman modes are impulsively and simultaneously excited, T-SRS features the natural-linewidth-limit spectral line shapes, laser-bandwidth-determined spectral range, and improved sensitivity. With ~150-fs laser pulses, we boost the sensitivity of typical Raman modes to the sub-mM level. With all-plane-mirror high-speed time-delay scanning, we further demonstrated hyperspectral SRS imaging of live-cell metabolism and high-density multiplexed imaging with the natural-linewidth-limit spectral resolution. T-SRS shall find valuable applications for advanced Raman imaging.

Transient Stimulated Raman Excited Fluorescence Spectroscopy.

The pursuit of better sensitivity has always been one of the central themes in Raman spectroscopy. Recently, all-far-field single-molecule Raman spectroscopy has been demonstrated by a novel hybrid spectroscopy that couples Raman scattering with fluorescence emission. However, such frequency-domain spectroscopy lacks efficient hyperspectral excitation methods and encounters intrinsic strong fluorescence backgrounds from electronic transitions, hindering its applications in advanced Raman spectroscopy and microscopy. Here we report the ultrafast time-domain spectroscopy counterpart named transient stimulated Raman excited fluorescence (T-SREF): excited by two successive broadband femtosecond pulse pairs (i.e., the pump and Stokes pulses) with time-delay scanning, strong vibrational wave packet interference is revealed on the time-domain fluorescence trace, resulting in background-free spectra of the corresponding Raman modes after the Fourier transform. T-SREF achieves background-free Raman spectra of electronic-coupled vibrational modes with sensitivity up to the level of a few molecules, which paves the way for supermultiplexed fluorescence detection and molecular dynamics sensing.

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence.

Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.

Chemical sectioning fluorescence tomography: high-throughput, high-contrast, multicolor, whole-brain imaging at subcellular resolution.

A thorough neuroanatomical study of the brain architecture is crucial for understanding its cellular compositions, connections, and working mechanisms. However, the fine- and multiscale features of neuron structures make it challenging for microscopic imaging, as it requires high contrast and high throughput simultaneously. Here, we propose chemical sectioning fluorescence tomography (CSFT) to solve this problem. By chemically switching OFF/ON the fluorescent state of the labeled proteins (FPs), we light only the top layer as thin as submicron for imaging without background interference. Combined with the wide-field fluorescence micro-optical sectioning tomography (fMOST) system, we have shown multicolor CSFT imaging. We also demonstrate mouse whole-brain imaging at the subcellular resolution, as well as the power for quantitative acquisition of synaptic-connection-related pyramidal dendritic spines and axon boutons on the brain-wide scale at the complete single-neuron level. We believe that the CSFT method would greatly facilitate our understanding of the brain-wide neuron networks.

Ultra-bright Raman dots for multiplexed optical imaging.

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental 'spectral crowding' of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active  nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.

Establishment and Verification of Multiaxis Fatigue Life Prediction Model.

A fatigue life prediction model with multiaxis load is proposed. The model introduces a new effective cyclic parameter, equivalent stress on the critical surface, to modify the Suntech model. The new damage parameters are not related to empirical constants, hence more applicable for practical application in engineering. The multiaxis fatigue test was carried out with high-strength aluminum alloy 7075-T651, and the multiaxis fatigue life prediction of the test piece was conducted with the finite element software. The experiment result shows that the model proposed is effective for predicting the fatigue life under multiaxis load.

Combining the best of two worlds: Stimulated Raman excited fluorescence.

The pursuit of a hybrid spectroscopy that combines the superb sensitivity of fluorescence and the high chemical specificity of Raman scattering has lasted for 40 years, with multiple experimental and theoretical attempts in the literature. It was only recently that the stimulated Raman excited fluorescence (SREF) process was successfully observed in a broad range of fluorophores. SREF allows single-molecule vibrational spectroscopy and imaging in the optical far field without relying on plasmonic enhancement. In this perspective, we will first review the historical efforts that lead to the successful excitation and detection of SREF, followed by the underlying physical principles, then the remaining technical challenges will be discussed, and, at last, the future opportunities in this old but yet newly emerged spectroscopy are outlined.

Strong Concentration Enhancement of Molecules at the Interface of Aqueous Microdroplets.

Water is arguably the most common and yet least understood material on Earth. The interface between water and a hydrophobic medium, such as air, oil, or lipids, plays a fundamental role in chemistry and biology. However, the behavior of molecules at interface of micron-sized water droplets (microdroplets) in such media is poorly characterized. Herein we employed two-photon fluorescence microscopy and Förster resonant energy transfer imaging to study the probe localization in water-oil microdroplets with high contrast and resolution. We found that there exists a general effect of surface enrichment and orientation alignment for water-soluble probes. Remarkably, probes are concentrated into a ∼10 nm thin layer at the microdroplet water-oil interface by up to 10 000-fold compared to the bulk counterpart. We suggest that the strong enrichment and alignment of water-soluble molecules, likely to be induced in part by a local electric field at the interface, could be a major factor accounting for orders of magnitude faster reaction rates observed in aqueous microdroplets compared to their bulk counterparts.

Strong Electric Field Observed at the Interface of Aqueous Microdroplets.

Chemical reactions in aqueous microdroplets often exhibit unusual kinetic and thermodynamic properties not observed in bulk solution. While an electric field has been implicated at the water interface, there has been no direct measurement in aqueous microdroplets, largely due to the lack of proper measurement tools. Herein, we employ newly developed stimulated Raman excited fluorescence microscopy to measure the electric field at the water-oil interface of microdroplets. As determined by the vibrational Stark effect of a nitrile-bearing fluorescent probe, the strength of the electric field is found to be on the order of 107 V/cm. This strong electric field aligns probe dipoles with respect to the interface. The formation of the electric field likely arises from charge separation caused by the adsorption of negative ions at the water-oil interface of microdroplets. We suggest that this strong electric field might account in part for the unique properties of chemical reactions reported in microdroplets.

Metabolic Activity Phenotyping of Single Cells with Multiplexed Vibrational Probes.

Quantitative measurements of metabolic activities of individual cells are essential to understanding questions in diverse fields in biology. To address this challenge, we present a method, termed metabolic activity phenotyping (MAP), to probe metabolic fluxes by utilizing multiplexed vibrational metabolic probes. With specifically designed single-whole-cell confocal micro-Raman spectroscopy, quantitative measurement of lipid and protein synthesis activity was achieved with high throughput (several orders of magnitude improvement over a commercial confocal system). In addition, metabolic heterogeneity upon various drug treatments was also revealed and evaluated at the single-cell level. We further demonstrated that MAP was more robust than the label-free Raman methods and was able to make the correct classification among diverse cancer types and breast cancer subtypes by exploring the dimension of metabolism. The capability of MAP to explore metabolic profiles at the single-cell level makes it a valuable tool for basic single-cell studies as well as other screening applications.

Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy.

Being able to image chemical bonds with high sensitivity and speed, stimulated Raman scattering (SRS) microscopy has made a major impact in biomedical optics. However, it is well known that the standard SRS microscopy suffers from various backgrounds, limiting the achievable contrast, quantification and sensitivity. While many frequency-modulation (FM) SRS schemes have been demonstrated to retrieve the sharp vibrational contrast, they often require customized laser systems and/or complicated laser pulse shaping or introduce additional noise, thereby hindering wide adoption. Herein we report a simple but robust strategy for FM-SRS microscopy based on a popular commercial laser system and regular optics. Harnessing self-phase modulation induced self-balanced spectral splitting of picosecond Stokes beam propagating in standard single-mode silica fibers, a high-performance FM-SRS system is constructed without introducing any additional signal noise. Our strategy enables adaptive spectral resolution for background-free SRS imaging of Raman modes with different linewidths. The generality of our method is demonstrated on a variety of Raman modes with effective suppressing of backgrounds including non-resonant cross phase modulation and electronic background from two-photon absorption or pump-probe process. As such, our method is promising to be adopted by the SRS microscopy community for background-free chemical imaging.

Stearic Acid Coated MgO Nanoplate Arrays as Effective Hydrophobic Films for Improving Corrosion Resistance of Mg-Based Metallic Glasses.

Mg-based metallic glasses (MGs) are widely studied due to their high elasticity and high strength originating from their amorphous nature. However, their further application in many potential fields is limited by poor corrosion resistance. In order to improve this property, an MgO nanoplate array layer is first constructed on the surface of Mg-based MGs by cyclic voltammetry (CV) treatments. In this situation, the corrosion resistance and hydrophilicity of the material are enhanced. Then, stearic acid (SA) can effectively adhere onto the surface of the MgO layer to form a superficial hydrophobic film with a water contact angle (WCA) of 131°. As a result, the SA coated MgO hydrophobic film improves the corrosion resistance of Mg-based MGs in 3.5 wt.% NaCl solution obviously. In addition, the effects of four technological parameters (solution concentration, sweep rate, cycle number, and reaction temperature) in the CV process on the morphology and size of nano-products are investigated in detail. The work proposes a new method for the creation of nanostructures on the surface of materials and provides a new idea to increase the corrosion resistance of MGs. The related method is expected to be applied in wider fields in future.

Electron-plasmon interaction on lithium niobate with gold nanolayer and its field distribution dependent modulation.

Surface plasmon resonance (SPR) enables strong field confinement, opening thereby new avenues for device miniaturization and reducing energy consumption. Plasmonic devices with electrical tunability attract tremendous interest for various applications. Most of the current researches achieved SPR modulation with relatively large driving voltages, or by other relatively low-speed tuning approaches, such as thermo-optic, magneto-optic, acousto-optic etc. In this paper, we propose and demonstrate an efficiently electrical SPR modulation based on lithium niobate (LN) with gold nanolayer (~81 nm) via electron-plasmon interaction. Efficient intensity modulation and wavelength shift (in visible band) of ~5.7 dB/V and ~36.3 nm/V are respectively obtained with low DC current. More importantly, modulation phenomenon of field distribution dependent is also observed and experimentally unveiled. Further performance is analyzed in terms of AC modulation and polarization characteristics. This key achievement opens up opportunities for applications such as optical interconnection, electric field sensing, electrically plasmonic modulation, etc.

Stimulated Raman Excited Fluorescence Spectroscopy of Visible Dyes.

Fluorescence spectroscopy and Raman spectroscopy are two major classes of spectroscopy methods in physical chemistry. Very recently, stimulated Raman excited fluorescence (SREF) has been demonstrated ( Xiong, H.; et al. Nature Photonics , 2019 , 13 , 412 - 417 ) as a new hybrid spectroscopy that combines the vibrational specificity of Raman spectroscopy with the superb sensitivity of fluorescence spectroscopy (down to the single-molecule level). However, this proof-of-concept study was limited by both the tunability of the commercial laser source and the availability of the excitable molecules in the near-infrared. As a result, the generality of SREF spectroscopy remains unaddressed, and the understanding of the critical electronic preresonance condition is lacking. In this work, we built a modified excitation source to explore SREF spectroscopy in the visible region. Harnessing a large palette of red dyes, we have systematically studied SREF spectroscopy on a dozen different cases with a fine spectral interval of several nanometers. The results not only establish the generality of SREF spectroscopy for a wide range of molecules but also reveal a tight window of proper electronic preresonance for the stimulated Raman pumping process. Our theoretical modeling and further experiments on newly synthesized dyes also support the obtained insights, which would be valuable in designing and optimizing future SREF experiments for single-molecule vibrational spectroscopy and supermultiplex vibrational imaging.

Fano Resonance on Nanostructured Lithium Niobate for Highly Efficient and Tunable Second Harmonic Generation.

Second harmonic generation (SHG) is an important nonlinear process which is critical for applications, such as optical integrated circuit, nonlinear microscopy, laser, etc. Many challenges remain in the improvement of nonlinear conversion efficiency, since the typical value is of only 10-5 in nanostructures. Here, we theoretically demonstrate a periodic structure consisting of a lithium niobate (LN) bar and an LN disk, on a nanoscale (~300 nm) thin-film platform, which is proposed for a highly efficient SHG. By breaking the structure symmetry, a Fano resonance with a high Q, up to 2350 and a strong optical field enhancement reaching forty-two folds is achieved, which yields a high conversion efficiency, up to 3.165 × 10-4. In addition to its strong second harmonic (SH) signal, we also demonstrate that by applying only 0.444 V on the planar electrode configurations of the nanostructured LN, the wavelength of SH can be tuned within a 1 nm range, while keeping its relatively high conversion efficiency. The proposed structure with the high nonlinear conversion efficiency can be potentially applied for a single-molecule fluorescence imaging, high-resolution nonlinear microscopy and active compact optical device.

Stimulated Raman Excited Fluorescence Spectroscopy and Imaging.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information1-3. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive3-5. Here we report a hybrid technique of Stimulated Raman Excited Fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the "color barrier" of fluorescence. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-sought-after goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SERF would be a valuable tool for chemistry and biology.

Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy.

Recently we have reported electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy as a powerful technique for super-multiplex imaging ( Wei, L. ; Nature 2017 , 544 , 465 - 470 ). However, under rigorous electronic resonance, background signal, which mainly originates from pump-probe process, overwhelms the desired vibrational signature of the chromophores. Here we demonstrate electronic resonant stimulated Raman scattering (er-SRS) microspectroscopy and imaging through suppression of electronic background and subsequent retrieval of vibrational peaks. We observed a change of the vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian as the electronic resonance was approached, in agreement with theoretical prediction. In addition, resonant Raman cross sections have been determined after power-dependence study as well as Raman excitation profile calculation. As large as 10-23 cm2 of resonance Raman cross section is estimated in er-SRS, which is about 100 times higher than previously reported in epr-SRS. These results of er-SRS microspectroscopy pave the way for the single-molecule Raman detection and ultrasensitive biological imaging.

Yucca fern shaped CuO nanowires on Cu foam for remitting capacity fading of Li-ion battery anodes.

To remit capacity fading of lithium ion battery (LIB) anodes, freestanding yucca fern shaped CuO nanowires (NWs) on Cu foams are fabricated as anodes by combining facile and scalable anodization of copper foams followed by calcination. The porous and radial configuration of the hierarchical CuO NWs on the Cu foam substrate guarantees the remarkably improved electrochemical performance with durable cycle stability and excellent rate capability compared with CuO NWs on Cu foils. The reversible capacity remains 461.5 mAh/g after 100 repeated cycles at a current density of 100 mA/g, and a capacity of 150.6 mAh/g even at a high rate of 1000 mA/g. By examining the surface morphology of the cycled samples, possible performance fading route is proposed. The 3D CuO NWs network with a porous architecture simutaneously reduces the ion diffusion distances, promotes the electrolyte permeation and electronic conductivity. This novel strategy might open a new window to develop durable CuO based composite anodes for LIBs.

Improvement of the mechanical properties and corrosion resistance of biodegradable ß-Ca3(PO4)2/Mg-Zn composites prepared by powder metallurgy: the adding ß-Ca3(PO4)2, hot extrusion and aging treatment.

In this study, 10%ß-Ca3(PO4)2/Mg-6%Zn (wt.%) composites with Mg-6%Zn alloy as control were prepared by powder metallurgy. After hot extrusion, the as-extruded composites were aged for 72h at 150°C. The effects of the adding ß-Ca3(PO4)2, hot extrusion and aging treatment on their microstructure, mechanical properties and corrosion resistance were investigated. The XRD results identified α-Mg, MgZn phase and ß-Ca3(PO4)2 phase in these composites. After hot extrusion, grains were significantly refined, and the larger-sized ß-Ca3(PO4)2 particles and coarse MgZn phases were broken into linear-distributed ß-Ca3(PO4)2 and MgZn phases along the extrusion direction. After aging treatment, the elements of Zn, Ca, P and O presented a more homogeneous distribution. The compressive strengths of the ß-Ca3(PO4)2/Mg-Zn composites were approximately double those of natural bone, and their densities and elastic moduli matched those of natural bone. The immersion tests and electrochemical tests revealed that the adding ß-Ca3(PO4)2, hot extrusion and aging treatment could promote the formation of protective corrosion product layer on the sample surface in Ringer's solution, which improved corrosion resistance of the ß-Ca3(PO4)2/Mg-Zn composites. The XRD results indicated that the corrosion product layer contained Mg(OH)2, ß-Ca3(PO4)2 and hydroxyapatite (HA). The cytotoxicity assessments showed the as-extruded ß-Ca3(PO4)2/Mg-Zn composite aged for 72h was harmless to L-929 cells. These results suggested that the ß-Ca3(PO4)2/Mg-Zn composites prepared by powder metallurgy were promising to be used for bone tissue engineering.