COHERENT ANTI-STOKES RAMAN SCATTERING MICROSCOPY TO MONITOR DRUG DISSOLUTION IN DIFFERENT ORAL PHARMACEUTICAL TABLETS

Coherent anti-Stokes Raman scattering(CARS)microscopy is used to visualize the release of a model drug(theophylline)from a lipid(tripalmitin)based tablet during dissolution.The effects of transformation and dissolution of the drug are imaged in real time.This study reveals that the manufacturing process causes significant differences in the release process:tablets prepared from powder show formation of theophylline monohydrate on the surface which prevents a controlled drug release,whereas solid lipid extrudates did not show formation of monohydrate.This visualization technique can aid future tablet design.

Supercontinuum fiber laser-based coherent anti-Stokes Raman scattering microscopy for label-free chemical imaging

Coherent anti-Stokes Raman scattering(CA RS)microscopy can resolve the chemical compo-nents and distribution of living biological systems in a label-firee manner and is favored in several disciplines.Current CA RS microscopes typically use bulky,high-performance solid-state lasers,which are expensive and sensitive to environmental changes.With their relatively low cost and environmental sensitivity,supercontinum fiber(SF)lasers with a small footprint have found increasing use in biomedical applications.Upon these features,in this paper,we homebuilt a low-cost CARS microscope based on a SF laser module(scCA RS microscope).This SF laser module is specially customized by adding a time synchronized seed source channel to the SF laser to form a dual-channel output laser.The performance of the scCARS microscope is evaluated with dimethyl sulfoxide,whose results confirm a spatial resolution of better than 500nm and a detection sensitivity of millimolar concentrations.The dual-color imaging capability is further demonstrated by imaging different species of mixed microspheres.We finally explore the potential of our scCARS microscope by mapping lipid droplets in different cancer cells and corneal stromal lenses.

Simultaneous measurements of global vibrational spectra and dephasing times of molecular vibrational modes by broadband time-resolved coherent anti-Stokes Raman scattering spectrography

In broadband coherent anti-Stokes Raman scattering （CARS） spectroscopy with supercontinuum （SC）, the simultaneously detectable spectral coverage is limited by the spectral continuity and the simultaneity of various spectral components of SC in an enough bandwidth. By numerical simulations, the optimal experimental conditions for improving the SC are obtained. The broadband time-resolved CARS spectrography based on the SC with required temporal and spectral distributions is realised. The global molecular vibrational spectrum with well suppressed nonresonant background noise can be obtained in a single measurement. At the same time, the measurements of dephasing times of various molecular vibrational modes can be conveniently achieved from intensities of a sequence of time-resolved CARS signals. It will be more helpful to provide a complete picture of molecular vibrations, and to exhibit a potential to understand not only both the solvent dynamics and the solute-solvent interactions, but also the mechanisms of chemical reactions in the fields of biology, chemistry and material science.

Selective excitation of molecular mode in a mixture by femtosecond resonance-enhanced coherent anti-Stokes Raman scattering spectroscopy

Femtosecond time-resolved coherent anti-Stokes Raman scattering （CARS） spectroscopy is used to investigate gaseous molecular dynamics. Due to the spectrally broad laser pulses, usually poorly resolved spectra result from this broad spectroscopy. However, it can be demonstrated that by the electronic resonance enhancement optimization control a selective excitation of specific vibrational mode is possible. Using an electronically resonance-enhanced effect, iodine molecule specific CARS spectroscopy can be obtained from a mixture of iodine-air at room temperature and a pressure of 1 atm （corresponding to a saturation iodine vapour as low as about 35 Pa）. The dynamics on either the electronically excited state or the ground state of iodine molecules obtained is consistent with previous studies （vacuum, heated and pure iodine） in the femtoseeond time resolved CARS spectroscopy, showing that an effective method of suppressing the non-resonant CARS background and other interferences is demonstrated.

Selective excitation and suppression of coherent anti-Stokes Raman scattering by shaping femtosecond pulses

Femtosecond coherent anti-Stokes Raman scattering （CARS） suffers from poor selectivity between neighbouring Raman levels due to the large bandwidth of the femtosecond pulses. This paper provides a new method to realize the selective excitation and suppression of femtosecond CARS by manipulating both the probe and pump （or Stokes） spectra. These theoretical results indicate that the CARS signals between neighbouring Raman levels are differentiated from their indistinguishable femtosecond CARS spectra by tailoring the probe spectrum, and then their selective excitation and suppression can be realized by supplementally manipulating the pump （or Stokes） spectrum with the π spectral phase step.

Coherent coupling between vibrational modes of C-H bonds at different positions studied by femtosecond time-resolved coherent anti-Stokes Raman scattering

We performed femtosecond time-resolved coherent anti-Stokes Raman scattering （fs-CARS） measurements on liquid toluene and PVK film. For both samples, we selectively excited the CH stretching vibrational modes and observed the expected quantum beat signals. The frequency of the well-defined beats is in good agreement with the energy difference between the two simultaneously excited modes, which demonstrates that a coherent coupling between the vibrational modes of the C H chemical bonds exists at the different positions of the molecules. The dephasing times of the excited modes are obtained simultaneously.

High-resolution imaging of graphene by tip-enhanced coherent anti-Stokes Raman scattering

Coherent anti-Stokes Raman scattering(CARS)is able to enhance molecular signals by vibrational coherence compared to weak Raman signal.The surface or tip enhancement are successful technologies,which make it possible for Raman to detect single molecule with nanometer resolution.However,due to technical diffculties,tip-enhanced CARS(TECARS)is not as successful as expected.For single molecular detection,high sensitivity and resolution are two main challenges.Here,we reported the first single atom layer TECARS imaging on Graphene with the highest resolution about 20 nm,which has ever been reported.The highest EF_(TECARS/CARS) is about 10^(4),the similar order of magnitude with SECARS(EF of tip is usually smaller than that of substrates).Such resolution and sensitivity is promising for medical,biology and chemical applications in the future.

Wide-Field Vibrational Phase Contrast Imaging Based on Coherent Anti-Stokes Raman Scattering Holography

We propose and implement a wide-field vibrational phase contrast detection to obtain imaging of imaginary components of third-order nonlinear susceptibility in a coherent anti-Stokes Raman scattering （CARS） microscope with full suppression of the non-resonant background. This technique is based on the unique ability of recovering the phase of the generated CARS signal based on holographic recording. By capturing the phase distributions of the generated CARS field from the sample and from the environment under resonant illumination, we demonstrate the retrieval of imaginary components in the CARS microscope and achieve background free coherent Raman imaging.

Coherent Raman scattering imaging of lipid metabolism in cancer

Cancer cells dysregulate lipid metabolism to accelerate energy production and biomolecule synthesis for rapid growth.Lipid metabolism is highly dynamic and intrinsically heterogeneous at the single cell level.Although°uorescence microscopy has been commonly used for cancer research,bulky°uorescent probes can hardly label small lipid molecules without perturbing their biological activities.Such a challenge can be overcome by coherent Raman scattering(CRS)microscopy,which is capable of chemically selective,highly sensitive,submicron resolution and high-speed imaging of lipid molecules in single live cells without any labeling.Recently developed hyperspectral and multiplex CRS microscopy enables quantitative mapping of various lipid metabolites in situ.Further incorporation of CRS microscopy with Raman tags greatly increases molecular selectivity based on the distinct Raman peaks well separated from the endogenous cellular background.Owing to these unique advantages,CRS microscopy sheds new insights into the role of lipid metabolism in cancer development and progression.This review focuses on the latest applications of CRS microscopy in the study of lipid metabolism in cancer.

Theoretical and experimental investigations of coherent phonon dynamics in sapphire crystal using femto-second time-resolved coherent anti-Stokes Raman scattering

We report on the theoretical and the experimental investigations of the coherent phonon dynamics in sapphire crystal using the femtosecond time-resolved coherent anti-Stokes Raman scattering （fs-CARS） technique. The temporal chirped white-light continuum （WLC） is used for the Stokes pulse, therefore we can perform the selective excitation of the phonon modes without using a complicated laser system. The expected quantum beat phenomenon is clearly observed. The theoretical formulas consist very well with the experimental results. The dephasing times of the excited phonon modes, the wavenumber difference, and the phase shift between the simultaneously excited modes are obtained and discussed. This work opens up a way to study directly high-frequency coherent phonon dynamics in bulk crystals on a femtosecond time scale and is especially helpful for understanding the nature of coherent phonons.

Analysis of detection limit to time-resolved coherent anti-Stokes Raman scattering nanoscopy

In the implementation of CARS nanoscopy, signal strength decreases with focal volume size decreasing. A crucial problem that remains to be solved is whether the reduced signal generated in the suppressed focal volume can be detected. Here reported is a theoretical analysis of detection limit （DL） to time-resolved CARS （T-CARS） nanoscopy based on our proposed additional probe-beam-induced phonon depletion （APIPD） method for the low concentration samples. In order to acquire a detailed shot-noise limited signal-to-noise （SNR） and the involved parameters to evaluate DL, the T-CARS process is described with full quantum theory to estimate the extreme power density levels of the pump and Stokes beams determined by saturation behavior of coherent phonons, which are both actually on the order of ~ 109 W/cm2. When the pump and Stokes intensities reach such values and the total intensity of the excitation beams arrives at a maximum tolerable by most biological samples in a certain suppressed focal volume （40-nm suppressed focal scale in APIPD method）, the DL correspondingly varies with exposure time, for example, DL values are 103 and 102 when exposure times are 20 ms and 200 ms respectively.

Selective Excitation of Two-pulse Femtosecond Coherent Anti-Stokes Raman Scattering in a Mixture

In this paper, we experimentally study the selective excitation of two-pulse femtosecond coherent anti-Stokes Raman scattering (CARS) in a mixture of dibromomethane (CH2Br2) and chloroform (CHCl3) by adaptive pulse shaping based on genetic algorithm. Second harmonic generation frequency-resolved optical gating (SHG-FROG) traces indicate that the spectral amplitude and phase of the optimal pulse are both modulated. Finally, we discuss the physical mechanism for the selective excitation of femtosecond CARS based on the retrieved information from SHG-FROG traces.

Stimulated Raman scattering microscopy on biological cellular machinery

Beneting from the developments of advanced optical microscopy techniques,the mysteries of biological functions at the cellular and subcellular levels have been continuously revealed.Stimulated Raman scattering(SRS)microscopy is a rapidly growing technique that has attracted broad attentions and become a powerful tool for biology and biomedicine,largely thanks to its chemical specicity,high sensitivity and fast image speed.This review paper introduces the principles of SRS,discusses the technical developments and implementations of SRS microscopy,then highlights and summarizes its applications on biological cellular machinery andnally shares our visions of potential breakthroughs in the future.

Computational coherent Raman scattering imaging:breaking physical barriers by fusion of advanced instrumentation and data science

Coherent Raman scattering(CRS)microscopy is a chemical imaging modality that provides contrast based on intrinsic biomolecular vibrations.To date,endeavors on instrumentation have advanced CRS into a powerful analytical tool for studies of cell functions and in situ clinical diagnosis.Nevertheless,the small cross-section of Raman scattering sets up a physical boundary for the design space of a CRS system,which trades off speed,signal fidelity and spectral bandwidth.The synergistic combination of instrumentation and computational approaches offers a way to break the trade-off.In this review,we first introduce coherent Raman scattering and recent instrumentation developments,then discuss current computational CRS imaging methods,including compressive micro-spectroscopy,computational volumetric imaging,as well as machine learning algorithms that improve system performance and decipher chemical information.We foresee a constant permeation of computational concepts and algorithms to push the capability boundary of CRS microscopy.

High enhancement factor of Au nano triangular prism structure for surface enhanced coherent anti-Stokes Raman scattering

Coherent anti-Stokes Raman scattering spectroscopy（CARS） is a well-known detecting tool in biosensing and nonlinear spectroscopy. It can provide a non-invasive alternative without the need for exogenous labels,while the enhancement factor for surface plasmon resonances（SPR） are extensively used to increase the local field close to the oscillators and which can obtain high enhancement. In this work, we investigate the enhancement factor of our structure for surface-enhanced coherent anti-Stokes Raman scattering. The absorption spectrum of the structure has been studied, a wide range of absorption has been realized. The enhancement can be as high as 10^（16） over standard CARS. Our design is very useful for improving the enhancement factor of surface-enhanced coherent anti-Stokes Raman scattering.

Nonlinear optical microscopy for skin in vivo:Basics,development and applications

Multi-photon microscopy(MPM)and coherent anti-Stokes Raman scattering(CARS)are two advanced nonlinear optical imaging techniques,which provide complementary information and have great potential in combination for noninvasive in vrito biomedical applications.This paper provides a detailed discussion of the basics,development and applications of these technologies for in vrivo skin research,covering the following topics:The principle and advantage of MPM and CARS,instrumentation development for in vino applications,MPM and CARS of normal skin,application of MPM and CARS in skin cancer and disease diagnosis;application of MPM in skin disease intervention,ie.,imaging guided two-photon photothermolysis.

Simultons in Nonstationary CARS by Polaritons: Energy and Velocity

This paper is the continuation of our previous research in which we studied such aspects of CARS spectroscopy in dipole-active crystals by polaritons as the regimes of coherent simultaneous propagation of three waves (anti-Stokes, Stokes, and the pump field) to increase the efficiency of CARS as a spectroscopic method. In our previous research, we have shown the possibility of the existence of simultons at all frequencies of interacting waves. All interacting waves were supposed to be linearly polarised and plane, the medium was assumed to be nonmagnetic, and the medium was transparent at frequencies of anti-Stokes, Stokes, and the pump field (laser). The purpose of the present paper is to consider the energy carried by electromagnetic waves and its relationship with the gain factor and velocity of the simultons.

相干反斯托克斯拉曼散射显微成像技术研究

基于全量子理论对相干反斯托克斯拉曼散射(CARS)过程进行了分析,在此基础上搭建了单频CARS显微成像系统,获得了不同尺寸聚苯乙烯微球高对比度的CARS显微图像.为了标定成像系统的空间分辨率,采用逐点扫描方式对直径为110 nm聚苯乙烯微球成像,从而重构出系统的点扩展函数.结果表明:该CARS显微成像系统的横向空间分辨率约为600 nm,而由阿贝衍射极限决定的理论空间分辨率约为300 nm.分析了导致分辨率降低的原因,并提出了解决方案.为实现纳米分辨的CARS显微成像打下了坚实的基础.

细胞内分子检测及成像技术研究

针对复杂生物学系统的研究和观测,指出对活体细胞内的分子及其相关事件,以高时空分辨率实现可视化、跟踪和定量处理,采用远场荧光成像是目前细胞内分子检测及成像的主要工具.在述评的基础上,介绍该课题组在活体细胞内分子检测及成像中的荧光显微成像和非标记检测技术的研究进展.围绕提高荧光显微成像分辨率,研究图像信息获取速率高的宽场成像方法,包括结构光照明和单分子定位显微.在结构光照明显微研究中,采用可编程的数字微镜器件代替需要精确移动的光栅对生物样品进行显微成像,获得了超分辨显微成像;在单分子定位显微成像研究中,搭建单分子定位显微成像系统,实测分辨率达到48nm,并获得了HeLa细胞突起中微丝束结构的纳米分辨图像;针对厚样品成像时存在的荧光串扰难题,提出利用双波长空间非相干光干涉照明,理论分析证明,其可实现厚度为35nm半高全宽的单一薄层的轴向选择性激发.在非标记检测成像技术方面,围绕相干反斯托克斯拉曼散射(coherentanti-Stokes Ramanscattering,CARS)方法中存在的问题展开研究.为获得完整的物质分子CARS光谱,利用飞秒激光脉冲泵浦PCF获得超连续谱激光输出同时作为泵浦光和斯托克斯光,实现超宽带时间分辨CARS光谱探测和显微成像技术;通过数值模拟获得优化超连续谱激光输出的时谱结构的实验条件,初步实现了满足实验所需的SC激光光源;通过调节探测光脉冲与SC激光脉冲之间的时间延迟实现时间分辨CARS,达到有效抑制非共振背景噪声,提高系统探测灵敏度的目的.利用超宽带时间分辨CARS光谱探测和显微成像技术,获得多种有机溶液在387～4092cm-1范围内,光谱分辨率达14cm-1的不同分子的CARS光谱信号;通过分析探讨实现超分辨CARS显微成像技术的途径,提出利用双探测光实现纳米分辨的方案.未来研究方向,在荧光显微方面,将结合双波长空间非相干光干涉照明和轴向纳米定位,实现完整细胞三维纳米分辨荧光成像,通过发展高量子效率的小分子荧光开关材料及高灵敏度高帧频的探测器,将单分子定位显微应用到活细胞三维纳米成像和分子追踪上;在非标记成像方面,将重点发展基于CARS原理的单分子检测和纳米成像方法,尤其是基于改进后的超连续谱的超宽带CARS光谱技术和纳米分辨的CARS显微技术.

CARS显微成像系统的空间分辨率标定

空间分辨率是衡量相干反斯托克斯拉曼散射(coherent anti-stokes Raman scattering,CARS)显微成像系统性能的一个重要指标.选择直径为110 nm的聚苯乙烯微球进行三维成像,根据各个点的位置数据与光谱数据形成的二维数组分别重构了系统x-y平面和x-z平面的点扩展函数,准确测试了自行搭建的CARS显微成像系统的横向空间分辨率约为484 nm,轴向空间分辨率约为3.17μm.