有机材料的双光子吸收物理特性及其应用

近年来，具有大双光子吸收截面的有机材料的物理性质及其应用的研究成为令人关注的重要研究课题。文章综述了有机材料的双光子吸收过程，物理特性以及其实验测试和研究方法，评述了有机材料的双光子吸收效应的应用，包括上转换散射，光学限幅，双光子荧光显微成像、三维光信息存储和光学微加工等。     

不同荧光波长的双光子共焦成像分析

研究了双光子共焦显微镜中不同荧光波长对成像特性的影响,导出了不同荧光波长的三维脉冲响应函数和三维光学传递函数并进行数值计算.研究结果表明：不同荧光波长对双光子共焦显微镜的三维光学传递函数、三维脉冲响应函数和空间截止频率产生明显的影响,随着荧光波长的增大,分辨率明显下降,但不会出现单光子共焦显微镜中的失锥现象,选取适当的荧光波长进行成像,有利于进一步改善图像分辨率和成像质量.     

双光子和多光子共焦显微镜的成像理论

对双光子和多光子共焦扫描显微镜的成像理论作了系统的理论分析,导出了双光子和多光子共焦显微镜成像系统的三维点扩散函数和三维光学传递函数,研究结果表明：双光子共焦显微镜比单光子共焦显微镜具有更高的横向分辨率和纵向分辨率,而多光子共焦扫描显微镜又比双光子共焦扫描显微镜具有更高的空间分辨率. 关键词：     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

双光子成像观测衰老对小鼠卵母细胞内DNA甲基转移酶表达的影响

量子点(QD)具有抗漂白能力强、量子产率高、激发谱宽和发射谱窄等优点,在生物荧光标记领域有较广泛的应用。利用QD 585和Hoechst 33342 (H342)双标记小鼠卵母细胞中DNA甲基转移酶(Dnmt)和染色体,通过双光子成像同时双通道探测它们的荧光图像,获得了Dnmt蛋白表达的三维空间分布。发现老龄小鼠卵母细胞不适合体外培养成熟：该成熟方式不仅引起老龄小鼠卵母细胞Dnmt蛋白含量的变化,而且还改变了它们在胞质中的空间分布。这些改变可能与异常的甲基化修饰有关。     

共焦扫描荧光显微镜的信噪比与信息量

在研制用于对厚的生物样品进行光学断层成像的共焦扫描荧光显微镜时，由于成像信号十分微弱及存在很强的多次散射作用，因此杂散光的抑制非常重要，而信噪比、信号背景比就成为决定能否获得高对比度、高分率图像的关键。运用光学信息量的概念，在已有的光学成像系统信息量计算、共焦扫描荧光显微镜信噪比及传递函数计算的基础上，详细分析了共焦扫描荧光显微镜信息量与信噪比等之间的定量关系。该关系表明，为了充分利用共焦扫描荧光显微镜的成像性能，必须选择适当的探测小孔。所得的结果对于共焦扫描荧光显微成像系统的研制有重要的实用价值。     

双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.     

荧光显微镜自组实验设计

利用光学支架、透镜、滤光片、平移台等在光学面包板上组装了荧光显微镜,组装仪器是集白光照明成像、激光激发荧光成像、荧光光谱探测等功能于一体的开放式系统.利用光学分辨率板白光照明条件下测量了荧光显微镜的空间分辨能力,采集了荧光样品CdTe/CdS量子点的荧光光谱和荧光图像.     

光子扫描隧道显微镜的光电成像系统研究

本文论述了光子扫描隧道显微镜（ＰＳＴＭ）的显微成像机理、成像规律，针对具体的物理模型进行数值模拟计算，并得到了与实际探测相一致的场分布规律。采用自行研制的光子扫描隧道显微镜（ＰＳＴＭ）的显微实验系统对多种样品进行了表面显微成像研究，获得了关于样品表面三维立体图像信息，通过多种图像处理手段对原始图像进行后期处理，得到了更具视觉效果、更为逼真的样品表面图像。     

多光子皮肤成像技术及其应用

多光子成像技术是一种层析能力好、信噪比高的新型光学成像技术。在皮肤光学三维检测中,多光子技术已经应用于无创在体成像,且已得到产业化开发。本文将首先介绍多光子皮肤检测系统的若干核心技术,即双光子自发荧光技术、二次谐波成像技术、荧光寿命成像技术、相干反斯托克斯-拉曼成像技术等,然后简要介绍多光子成像系统在皮肤疾病成像检测上的应用,最后分析该系统的优势和未来可能的发展趋势。     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

Compact two-photon fluorescence microscope based on a single-mode fiber coupler

We present a two-photon fluorescence microscope based on a three-port single-mode optical fiber coupler. It is found that the coupler behaves as a low-pass filter that can deliver an ultrashort-pulsed laser beam of as much as 150 mW of power in the wavelength range from 770 to 870 nm as well as collect a two-photon fluorescence signal in the visible range. As a result of using the fiber coupler, the new two-photon imaging system exhibts a number of advantages, including a compact arrangement, freedom from vibration from lasers and electronic devices, self-alignment, reduction of multiple scattering, and an enhanced optical sectioning effect. The effectiveness of the new instrument is demonstrated with a set of three-dimensional images of biological samples. This instrument may make two-photon fluorescence endoscopy possible for in vivo medical applications.     

Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy

Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, in reflection and transmission respectively, and the simultaneous nature of the acquisition of the two images allows pure TPF and SHG images in precise registration to be obtained.     

生物样品的散射和吸收特性对双光子激发和共焦成象深度的影响

本文研究了生物样品的散射和吸收特性对双光子激发和共焦成象深度的影响.生物组织内30μm的成象结果表明,双光子激发具有比共焦成象更深的成象深度.来自生物组织的模型实验和蒙特卡罗模拟均表明,散射和吸收的影响是导致这一特点的重要原因.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Non-descanned versus descanned epifluorescence collection in two-photon microscopy: Experiments and Monte Carlo simulations

We report on the design, test and Monte Carlo simulations of a non-descanned (NDS) collection port that we compare to a descanned (DS) port implemented on the same confocal microscope to carry out two-photon excitation fluorescence (TPEF) imaging. Our optical concept provides compactness, a wide field of view to the NDS port and allows the usage of small-area photosensors. The collection efficiency of the NDS port was measured with respect to those of the DS port as function of the imaging depth within a tissue-like optical phantom, for two high numerical aperture objectives. A NDS-to-DS collection ratio as high as about 30 was found for an imaging depth of 500 μm, corresponding to four mean scattering paths of the collected photons within the turbid medium. Measurements were fully interpreted by Monte Carlo simulations of light scattering through the turbid medium and collection by the spatio-angular apertured DS and NDS ports. Comparison of XZ cross-sectional views of mice liver samples imaged with the two ports emphasized the advantage of our NDS device for imaging deeply inside biological samples using TPEF microscopy.     

双光子激发荧光成像技术对小鼠植入前胚胎的实时观测

双光子激发的蓝移效应可使利用同一束超快激光同时激发多种不同荧光特性的生物荧光染料的设想得以实现。选取锁模飞秒掺钛蓝宝石(Ti-sapphire)激光器输出的730nm激光,分别激发Hoechst33342,Fluo-4,PI和Indo-1四种常用生物荧光染料,分别利用(455±15)nm,(540±15)nm,(580±16)nm和(500±15)nm四种滤光片获得特异性荧光图像。结合双光子激发荧光成像技术穿透深,光损伤小,信噪比好等优势,选取合适的荧光染料组,应用单束激光激发、双染双通道成像方法,对小鼠植入前胚胎内细胞中的钙信号和染色体进行三维、四维实时成像,为探究小鼠植入前胚胎发育规律提供一种全新的多参数观测手段。     

Three-dimensional in vivo scanning microscopy with inertia-free focus control

The acquisition of high-resolution images in three dimensions is of utmost importance for the morphological and functional investigation of biological tissues. Here, we present a laser scanning two-photon microscope with remote and motionless control of the focus position. The movement of the excitation spot along the propagation direction is achieved by shaping the laser wavefront with a spatial light modulator. Depending on the optical properties of the objective in use, this approach allows z movements in a range of tens to hundreds of micrometers with small changes of the point spread function. We applied this technique for the three-dimensional (3D) imaging of fluorescent cells in the mouse neocortex in vivo. The presented system bypasses the limitations of microscopes based on moving objectives, enabling high-resolution inertia-free 3D imaging.     

Acetylene-Substituted Two-Photon Absorbing Molecules With Rigid Elongated Pi-Conjugation: Synthesis,Spectroscopic Properties and Two-Photon Fluorescence Cell Imaging Applications

Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores for two-photon microscopic imaging. These molecules consist of a typical D–π–A structure, have different donors (D), the same π-conjugated center (π) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon (linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging.