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.     

Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.     

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.     

Membrane potential detection with second-harmonic generation and two-photon excited fluorescence: A theoretical comparison

We theoretically compare the performance of TPEF and SHG microscopy for membrane potential imaging. We argue that electrochromic TPEF and SHG membrane potential responses are reflections of the same phenomenon, and can be described in a unified manner as resulting from the linear Stark effect. We also show that TPEF and SHG exhibit similar sensitivities in the case of both electrochromic and orientational response mechanisms. Despite their similar sensitivities, SHG nevertheless presents advantages over TPEF for membrane potential imaging because of its remarkable spatial and spectral contrast, and because of its insensitivity to non-radiative excited-state damping mechanisms.     

Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy

We demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics.     

In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non-linear phenomena

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image contrast modes on the same microscope. Three-dimensional (3D) reconstructions of TPEF, SHG and THG images were also performed. Moreover, THG imaging technique has been tested as a potential, novel, non-destructive diagnostic tool for monitoring cellular processes in vivo, such as neuronal degeneration.     

Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH₂-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH₂ bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin’s autofluorescence and SFG signals arising from collagen fibrils’ non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.     

Nonlinear spectral imaging of human normal skin,basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.     

Second-harmonic generation as a DNA malignancy indicator of prostate glandular epithelial cells

This paper first demonstrates second-harmonic generation (SHG) in the intact cell nucleus, which acts as an optical indicator of DNA malignancy in prostate glandular epithelial cells. Within a scanning region of $2.7~\mu$m$\times 2.7~\mu$m in cell nuclei, SHG signals produced from benign prostatic hyperplasia (BPH) and prostate carcinoma (PC) tissues (mouse model C57BL/6) have been investigated. Statistical analyses ($t$ test) of a total of 405 measurements (204 nuclei from BPH and 201 nuclei from PC) show that SHG signals from BPH and PC have a distinct difference ($p < 0.05)$, suggesting a potential optical method of revealing very early malignancy in prostate glandular epithelial cells based upon induced biochemical and/or biophysical modifications in DNA.     

Molecular engineering of NLO-phores for new NLO microscopies

Novel microscopies based on nonlinear optical (NLO) phenomena such as two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) have gained overwhelming popularity in the biology community owing to the many advantages they provide in biological imaging. Examples of molecular engineering approaches toward NLO-probes specifically designed for SHG and/or TPEF imaging of lipid membranes and biological cells are given here, providing an illustration of their intriguing potential in the area of real-time, non-damaging imaging of biological structures. Optimized NLO-markers open new routes for improved monitoring and better understanding of fundamental dynamic processes. To cite this article: M. Blanchard-Desce, C. R. Physique 3 (2002) 439–448.     

肿瘤细胞DNA样品基于二次谐波发生(SHG)的非线性光谱学成像

二次谐波的产生是一个二阶非线性光学过程,这个过程只发生在中心对称系数不为零的非中心对称区域。利用二次谐波显微技术可以对各种具有内源性信号的生物组织进行无损伤实时成像,如结缔组织的胶原纤维或肌细胞的肌动球蛋白。在生物化学和结构生物学中,虽然DNA是由脱氧核糖核苷酸构成而蛋白质是由氨基酸残基组成,但是DNA和蛋白质大分子高级结构的形成机制是相似的。利用光谱学成像技术对不同DNA样品进行检测,获取DNA样品的SHG信号并进行高解析度成像。这些DNA样品包括基因组DNA溶液、细胞核提取物以及培养细胞的细胞核。实验结果表明在常规条件下可以获得基因组DNA溶液和细胞核提取物的SHG信号,但几乎观测不到来自培养细胞核区的SHG信号。通过在培养基中添加少量无水乙醇(体积比小于5%),可以在培养细胞的细胞核区域检测到SHG信号。推测在培养细胞中乙醇和DNA相互作用引起DNA分子构象发生变化,这些变化可能导致了DNA分子非线性光学性质的改变。     

多色双光子激发荧光显微技术实验研究

双光子激发荧光(two-photon excited fluorescence, TPEF)显微是一种非线性光学显微技术, 具有高的时间分辨率和空间分辨率、高的信噪比和固有的三维层析分辨能力等优点. 传统的TPEF显微一般采用波长可调谐的超短脉冲激光器作为光源. 在实际应用中, 利用TPEF显微技术研究含有多种荧光团或未知成分的待测样品, 往往需要多次改变激发光的波长以获得对各种荧光团的最佳激发. 为了同时获取不同荧光团的荧光信号, 利用超连续谱激光光源实现了多色TPEF显微成像, 实验中无需调节波长, 能够同时获得具有两种不同发射波长的荧光标记的铃兰根茎切片样品的TPEF图像. 实验结果表明, 与传统的TPEF显微相比, 该方法能够同时获取含有多种荧光团的待测样品的高对比度TPEF图像, 具有系统结构简单、操作简便、信息量大等优点, 在生物医学和材料科学等领域具有广阔的应用前景.     

Sensing cell metabolism by time-resolved autofluorescence

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.     

Thermo-optical switching enhanced with second harmonic generation in microring resonators

We investigate the thermo-optic effects on second harmonic generation (SHG) in AlGaAs/AlO(x) microring (MR) waveguides. We show that SHG can be utilized to enhance the thermal sensitivity of MRs. This sensitivity makes this structure appropriate for thermal modulation and switching of optical signals or temperature sensing.     

Single-beam and enhanced two-beam second-harmonic generation from silicon nanocrystals by use of spatially inhomogeneous femtosecond pulses

Optical second-harmonic generation (SHG) is used as a noninvasive probe of the interfaces of Si nanocrystals (NCs) embedded uniformly in an SiO2 matrix. Measurements of the generated SH mode verify that the second-harmonic polarization has a nonlocal dipole form proportional to (E x Delta inverted) E that depends on inhomogeneities in the incident field E, as proposed in recent models based on a locally noncentrosymmetric dipolar response averaged over the spherical NC interfaces. A two-beam SHG geometry is found to enhance this polarization greatly compared to single-beam SHG, yielding strong signals useful for scanning, spectroscopy, and real-time monitoring. This configuration provides a general strategy for enhancing the second-order nonlinear response of centrosymmetric samples, as demonstrated here for both Si nanocomposites and their glass substrates.     

超高真空中C60光聚合效应光学二次谐波的研究

利用１．０６４μｍ基频超短脉冲激光，使用光学二次谐波方法研究了超高真空中蒸镀在多晶银表面上的Ｃ６０薄膜紫外光照射下的光聚合现象，发现聚合以后的Ｃ６０二阶非线性响应增强，同时观察到光聚合的饱和效应，Ｃ６０光聚合二阶非线性的提高可以用电四极了磁偶极子对二次谐波贡献的增强来解释。     

Voigt位型下电介质/反铁磁/电介质结构二次谐波生成非倒易性研究

利用非线性传递矩阵方法研究了Voigt位型下电介质/反铁磁/电介质 结构二次谐波生成的非倒易性. 研究发现外加静磁场反向和电介质层排序翻转均对二次谐波输出产生影响, 出现了二次谐波生成的非倒易性. 二次谐波生成非倒易性频率区域在反铁磁共振区, 此区间正处于THz频段. 随着入射角度的增加, 非倒易性的效果越来越明显. 研究二次谐波生成的非倒易性, 可为反铁磁器件的设计加工提供理论支持.     

The temperature dependence of surface second-harmonic generation from the air-water interface

An investigation of the polarization and temperature dependence of surface second-harmonic generation (SHG) at the air-water interface shows that the intensity of the SHG signal decreases over the range 20–70°C. Accounting for the changes in the SHG polarization behaviour with temperature to extract the relative temperature dependence of X_zzz shows that it is similar to that obtained from simulation studies. The role of the interfacial refractive index in the interpretation of the SHG signals is addressed.     

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.