Examination of Validity of Paraxial Approximation in Second Harmonic Generation Microscopy under Low Numerical Aperture

By using a vectorial approach, the validity of paraxial approximation in second harmonic generation （SHG） microscopy under low numerical aperture （NA） is examined when the sample is a collagen fibril. Due to the larger value of dzzz and tensorial nature of SHG, the component Ez of the focused fieM may have strong effect on the radiation pattern of SHG. Numerical results indicate that when the value of NA exceeds 0.3, the effect of Ez can not be neglected, which results in the invalidation of paraxial approximation in SHG microscopy despite the fact that SHG microscopy is still under low NA focusing.     

A quasi-crystal model of collagen microstructure based on SHG microscopy

<正>Second harmonic generation(SHG) results from molecules which are polarized by an external electric field often provided by an intense laser beam.The polarizability depends on firstly the intrinsic structural properties of the substance and hence the second-order nonlinear susceptibility,and secondly the intensity and polarization direction of the incident light.The polarization characteristics of the beam are therefore of interest.In this letter,we discuss some considerations in SHG microscopy of collagen when the incoming beam is circularly polarized,and present some supporting results as well as a numerical analysis.We propose a quasi-crystal model of collagen microstructure in an effort to further our understanding on this protein.     

Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy

The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25 degrees-60 degrees C. We find that the SHG signal decreases rapidly starting at 45 degrees C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57 degrees C and worsens with increasing temperature. At 57 degrees C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60 degrees C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.     

晶体球中非相位匹配二次谐波产生及最佳聚焦条件

晶体球法是近年来在国际上发展起来的一种测量非线性系数的新方法。根据聚焦高斯光束二次谐波产生的孔径方程理论,分析了晶体球中的第Ⅰ类非相位匹配二次谐波产生过程,讨论了晶体球中第Ⅰ类非相位匹配二次谐波最佳聚焦参数的选择。报道了LiNbO3晶体球中皮秒激光脉冲抽运的相位匹配二次谐波实验。所得结果与理论预计相符。     

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.     

Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.     

Second-harmonic generation in the interior of an isotropic medium with quadratic nonlinearity by a focused inhomogeneously polarized pump beam

This paper presents a theoretical study of second-harmonic generation (SHG) by a focused pump beam in the interior of an isotropic medium, experimentally observed earlier. It shows that the spatial dispersion of the quadratic optical response of the substance can be responsible for this nonlinear process even when a macroscopic inversion center is present in the medium. It is established that this effect, which is impossible in the plane-wave approximation, also does not occur when a Gaussian pump beam with uniform distribution of the polarization state of the wave field over the cross section is used, but that the presence in the pump beam of higher transverse modes with polarization different from the fundamental mode can cause an SHG signal to appear. The conditions for a wave to appear at the doubled frequency are found, analytical dependences for its electric field and total power on the propagation coordinate, the degree of focusing, and the other parameters of the problem are obtained, and the requirements on the optimum experimental geometry are formulated. The dependence of the signal-beam power on the wavevector detuning is studied, and it is shown that interference effects can cause the wave at the second harmonic to disappear when a normal dispersion law is obeyed in the region between the fundamental and doubled frequencies. Zh. éksp. Teor. Fiz. 113, 1261–1276 (April 1998)     

Optical second-harmonic generation of Janus MoSSe monolayer

The transition metal dichalcogenides (TMD) monolayers have shown strong second-harmonic generation (SHG) owing to their lack of inversion symmetry. These ultrathin layers then serve as the frequency converters that can be intergraded on a chip. Here, taking MoSSe as an example, we report the first detailed experimental study of the SHG of Janus TMD monolayer, in which the transition metal layer is sandwiched by the two distinct chalcogen layers. It is shown that the SHG effectively arises from an in-plane second-harmonic polarization under paraxial focusing and detection. Based on this, the orientation-resolved SHG spectroscopy is realized to readily determine the zigzag and armchair axes of the Janus crystal with an accuracy better than ±0.6°. Moreover, the SHG intensity is wavelength-dependent and can be greatly enhanced (～ 60 times) when the two-photon transition is resonant with the C-exciton state. Our findings uncover the SHG properties of Janus MoSSe monolayer, therefore lay the basis for its integrated frequency-doubling applications.     

Sensing and compensation of femtosecond waveform distortion induced by all-order polarization mode dispersion at selected polarization states

We demonstrate full characterization of femtosecond pulse distortion induced by all-order polarization mode dispersion (PMD) at selected polarization states via second-harmonic generation (SHG) frequency-resolved optical gating (FROG) measurements at an average power of under 28 nW. By applying the inverse of the measured spectral phase via a programmable pulse shaper, we compress the distorted pulses from more than 3 ps to nearly bandwidth-limited durations of less than 500 fs. Our results show that SHG FROG measurements performed by using fiber-pigtailed aperiodically poled lithium niobate waveguides can serve as a robust and sensitive tool for characterization of PMD-induced spectral phase.     

Visualization of collagen regeneration in mouse dorsal skin using second harmonic generation microscopy

The purpose of this study is to highlight a clearer understanding of the process of collagen regeneration during wound healing. By means of second harmonic generation (SHG) microscopy, the changes of collagen arrangement at the wound margin were analyzed at 0, 3, 5, 7, 11 and 13 days post injury. The degree of collagen disorders associated with the healing process was quantitatively obtained using the aspect ratio of polar plot image of collagen azimuthal angles and the healing status of collagen could be estimated by arithmetical mean deviation (Ra) of the collagen SHG images. Our results suggest that SHG microscopy has potential advances in the collagen studies during wound healing and the arrangement of collagen fibers gradually transformed from disorder to order so as to contract the wound. It is capable of promoting clinical application of the noninvasive imaging tool and the analysis methods of collagen disorder as an effective scar management for prevention and treatment about aberrant healing. Original Text ? Astro, Ltd., 2009. The article is published in the original.     

Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology

We present a heuristic treatment which relates SHG image intensities, signal directionality, and observed morphology to the physical structure of collagen and cellulose fibrillar tissues. The SHG creation model is based upon relaxed phase matching conditions which account for dispersion, randomness, and axial momentum contributions from the media, and includes a mathematical treatment which relates SHG conversion efficiency to fibril diameter and packing through the inclusion of potential intensity amplification resultant from quasi-phase matching (QPM). A direct consequence of this theory is that SHG in biological tissues is not strictly a coherent process, and that the forward directed SHG has a longer coherence length than the backward component, Through this treatment, we show that the emission directionality and also conversion efficiency do not arise solely from the fibril size but also depend on packing density and order of the inter-fibril structure. We demonstrate these principles in comparing the SHG response in normal and Osteogenesis Imperfecta (OI) skin. We show that the observed directionality and decreased relative intensity in the diseased state is consistent with phase matching conditions arising from the decreased fibril size and more random assembly. We further use this theory to explain the differences in morphology seen in forward and backward collected SHG in fibrillar tissues (e.g., collagenous and cellulosic). Specifically, we attribute segmented appearance to destructive interference between small fibrils separated by less than the coherence length. We suggest the approach based on relaxed phasematching conditions is general in predicting the SHG response in tissues and may be broadly applicable in interpreting the SHG contrast for diagnostic applications.     

有机环形光纤中二次谐波产生的研究

本文从经典电磁理论出发，分析了有机环形光纤中二次谐波产生（ＳＨＧ）的转换效率，着重讨论利用环形光纤中模式的色散特性实现相位匹配等问题。报道了采用ＬＢ膜方法，制成半花菁环形光纤，由高灵敏度光检测计测出在强泵光（１．０６μｍ，ＹＡＧ激光）作用下该光纤中的二次谐波产生的实验结果。     

消荧光现象与表面谐波产生

消荧光现象是激光与物质表面非线性相互作用的结果，这是一种普遍现象。在表面谐波产生的研究中，它常常被忽略。大量研究表明：表面谐波产生与消荧光现象，有着密切的内在联系。本文以消荧光观点详细讨论了表面谐波产生的机制，提出了表面谐波产生的消荧光模型。新机制可归纳为：（ａ）多光子受激吸收产生消荧光光斑，（ｂ）激光热效应等促进自发辐射转向准受激辐射，（ｃ）伴随荧光消失，部分产生荧光的激光能量转换为产生表面谐波     

Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging

We performed multiphoton fluorescence (MF) and second-harmonic generation (SHG) imaging on human basal cell carcinoma samples. In the dermis, basal cell carcinomas can be identified by masses of autofluorescent cells with relatively large nuclei and marked peripheral palisading. In the normal dermis, SHG from dermal collagen contributes largely to the multiphoton signal. However, within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals, indicating that normal collagen structures responsible for SHG have been altered. To better delineate the cancer cells and cancer stroma from the normal dermis, a quantitative MF to SHG index is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from the normal dermis. Our work shows that MF and SHG imaging can be an alternative for Mohs' surgery in the real-time guidance of the secure removal of basal cell carcinoma.     

Automatically tunable second-harmonic generation of dye lasers

A new technique which realizes the phase-matching of second harmonic generation (SHG) over a wide spectral range without mechanical or temperature tuning of the SHG crystal is developed. The phase-matching is achieved by changing the angle of the laser beam incident on the SHG crystal using dispersive components such as gratings or prisms. Over the entire spectral range of a Rhodamine-6G dye laser, the phase-matching is realized with a KDP crystal using this technique. This system is capable of producing tunable coherent ultraviolet light from 2300 Å to 3500 Å with an extremely fast scanning rate, if the dispersion of the prism and the SHG crystal is carefully matched.     

有机分子膜中各向异性的微扰理论和实验研究

采用微扰方法,对有机分子膜中分子规则取向所导致的各向异性进行了研究.给出了有机分子膜的二次谐波强度与基频光和倍频光的偏振方向,以及基板的拉膜方向之间关系的理论表达式,并对常见的几种组态进行了讨论和实验研究.理论计算和实验结果相一致. 关键词：     

Monitoring process of human keloid formation based on second harmonic generation imaging

In this paper, the morphological variation of collagen among the whole dermis from keloid tissue was investigated using second harmonic generation (SHG) microscopy. In the deep dermis of keloids, collagen bundles show apparently regular gap. In the middle dermis, the collagen bundles are randomly oriented and loosely arranged in the pattern of fine mesh while the collagen bundles are organized in a parallel manner in the superficial dermis near the epidermis. The developed parameters COI and BD can be used to further quantitatively describe these changes. Our results demonstrate the potential of SHG microscopy to understand the formation process of human keloid scar at the cellular level through imaging collagen variations in different depth of dermis.     

聚焦光场激发下中心对称球形粒子的二次谐波产生

在聚焦光场矢量衍射理论的基础上,利用贝塞尔函数的微分递推关系,推导了聚焦光场的梯度变化表达式。根据中心对称材料二次谐波产生的唯象模型,研究了单个中心对称球形纳米颗粒在聚焦光场激发下的二次谐波产生,考察了表面响应和体效应产生的二次谐波辐射分布随着数值孔径增大的变化规律,并利用聚焦光场及其梯度场分布特性讨论差异产生的原因。研究结果表明,激发光场的聚焦程度对体响应的影响更为显著。     

Demonstration of high conversion efficiency to second harmonic in a wide tuning range

This article discusses the second-harmonic generation (SHG) of tightly focused fs laser pulses in a BBO crystal. It was found theoretically and demonstrated experimentally that the spatial walk-off (SWO) and group-velocity dispersion (GVD) effects can suppress the pulse distortion and gain depression of SHG due to group-velocity mismatch (GVMM), thus keeping a reasonable pulse waveform and conversion efficiency simultaneously in a relatively wide tuning range. Using this approach, the SHG efficiency of a 2 mm long BBO crystal in a reflecting focusing geometry was enhanced significantly.     

Generation of tunable 7-fs ultraviolet pulses: achromatic phase matching and chirp management

Ultraviolet pulses with a duration of 7 fs are efficiently generated by frequency doubling the output of a noncollinear optical parametric amplifier. The ultraviolet pulses are tunable between 275 to 335 nm. The acceptance bandwidth of the doubling crystal is increased by a factor of 80 through high-order achromatic phase matching. The chirp of the visible pulses and the dispersion introduced along the beam path are compensated partially before and partially after the doubling crystal. For the design of the dispersion management, we investigate the second harmonic generation of pulses with mixed orders of chirp and explicitly discuss the transfer of the spectral phase in frequency doubling. A simple analytic theory is derived that correctly describes frequently observed spectral narrowing effects. We find that chirped SHG avoids spectral narrowing and allows for precompression of dispersion encountered in the ultraviolet beam path. We apply chirped SHG to generate 18.7 fs ultraviolet pulses in an extremely simple setup. PACS 42.65.Re; 42.65.Ky; 42.65.Yj