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：     

双光子技术在三维成像和三维存储技术中的应用

对双光子过程的空间分辨能力进行了理论分析。阐明了双光子技术在三维成像和三维存储中的独特优势。着重介绍了双光子技术与扫描共焦显微术、近场显微术相结合进行三维成像 ,以及一种多焦点多光子显微术和用连续光源激发的双光子三维成像技术的研究和进展情况。对建立在共焦显微镜基础之上的双光子三维光存储和微细加工方面的研究也作了回顾与展望     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

空间低频光共焦扫描法透过散射介质成象

本文使用4-F光学系统实现低通空间频率滤波,采用空间低频光共焦扫描法透过散射介质成象,实验表明,光路中加入简单的空间滤波系统后,明显改善共焦扫描法的成象质量和分辨率。     

超短光脉冲的平均功率和脉宽及锁模状态对双光子显微镜成象的影响

显微镜成象是研究生物活体细胞结构及活动规律的重要技术.飞秒光脉冲是多光子显微镜的主要光源,它的性能(脉宽、平均功率和锁模状态等)严重影响成象的质量.双光子激发荧光强度正比于超短脉冲平均功率的平方,反比于脉宽,锁模稳定状态严重影响成象的清晰度.对用Cameleon转染的HeLa细胞和GFP转基因水稻叶的实验研究结果与理论一致.     

应用光学——光学系统设计指南 第十二章 大气光学(续)

12.4 通过大气的成象在第12.2节中讨论过了辐射通过大气时的吸收和散射。在本节中我们分析吸收和散射对成象的影响。 12.4.1 亮度的传播就成象而论,吸收效应只不过是一种成象通量的衰减,这种衰减在任一波长上都是随距离成指数形式衰减的。然而,散射有双     

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.     

多光子成象术中深层图象损耗恢复的研究

本文在分析混浊介质对共焦三维扫描图象衰减的基础上,建立了多光子激发荧光扫描的深层图象恢复的数学模型.依照此模型对深层图象进行依赖组织光学参量的恢复,并给出了猪表皮三维扫描成象的恢复图象.     

一种新的生物医学用快速实时低相干显微成象原理

本文讨论了一种将共焦显微术与迈克耳逊干涉术相结合,并利用宽带低相干光源相干长度短的特点而形成的一种可对高密度非透明样品进行显微成象的方法,并将这种显微成象方法与共焦显微成象方法进行了比较,最后讨论了一种快速实时成象的原理,基于这种原理设计的仪器将为生物和医学工作者提供一种新的非侵入测量和诊断手段.     

共焦扫描显微成象的光学断层平面分辨率与信噪比之间的关系

在Fried的一维分辨度理论的基础上,系统地讨论了光学成象系统两维分辨率与信噪比之间的定量关系,并以反射式共焦显微成象为例,建立了实际显微成象系统分辨率的定量计算方法.所得出的结果对于选择共焦扫描显微成象系统的最佳参量及评价所设计的显微成象系统的性能具有重要的意义.     

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.     

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.     

Noninvasive determination of tissue optical properties based on radiative transfer theory

We present a feasibility study of a new method for determining the tissue optical properties, including the absorption and scattering coefficients and the scattering asymmetry factor. A state-of-the-art radiative transfer model for the coupled air/tissue system, based on rigorous radiative transfer theory, is used in our forward modeling simulations. The concept of the effective photon penetration depth is introduced and used to help determine the depth below, which information about the tissue will not be available through noninvasive imaging of a biological tissue using reflected diffuse light. Simulation results show that for accurate determination of tissue optical properties, one can use radiative transfer theory in conjunction with measurements of reflected radiances as well as other existing techniques.     

Ultrafast carrier dynamics of Cu2O thin film induced by two-photon excitation

Cuprous oxide (Cu₂O) has attracted plenty of attention for potential nonlinear photonic applications due to its superior third-order nonlinear optical property such as two-photon absorption. In this paper, we investigated the two-photon excitation induced carrier dynamics of a Cu₂O thin film prepared by radio-frequency magnetron sputtering, using the femtosecond transient absorption experiments. Biexponential dynamics including an ultrafast carrier scattering (< 1 ps) followed by a carrier recombination (> 50 ps) were observed. The time constant of carrier scattering under two-photon excitation is larger than that under one-photon excitation, due to the different transition selection rules and smaller absorption coefficient of the two-photon excitation.     

Two-photon optical-beam-induced current imaging of indium gallium nitride blue light-emitting diodes

Epilayers of packaged indium gallium nitride light-emitting diodes (LED's) are characterized by optical-beam-induced current (OBIC) and photoluminescence laser-scanning microscopy through two-photon excitation. Light scattering and absorption in the packaging material and the p-doped top layer of the LED's are greatly reduced as a result of employing a longer excitation wavelength, with energy that is less than the bandgap of the top p layer. Compared with single-photon OBIC, two-photon OBIC imaging not only exhibits superior image quality but also reveals more clearly the characteristics of the epilayers that are being focused on.     

Strong two-photon-induced fluorescence from a highly soluble polythiophene

Two-photon-induced fluorescence from a soluble polythiophene containing urethane side groups has been investigated using femto-second laser pulses at 800 nm. Strong two-photon fluorescence was measured in polymer solution. The quadratic dependence of the fluorescence on the excitation laser intensity confirmed the two-photon process. The measured two-photon absorption cross-section is larger as compared to those of other reported polythiophenes. This polymer can be readily hydrolyzed to yield a water soluble polythiophene which could be useful in biological imaging.     

Investigation of transient two-photon excited fluorescence in biological tissue

The fluorescence power from biological tissue excited by a femtosecond laser pulse compared with excitation power does not appear to obey a simple quadratic relationship given by the steady non-linear theory.A more reliable analysis is developed based on transient two-photon absorption because the response time of two-photon absorption is longer than the width of a femtosecond pulse.Good agreement is obtained between the theoretical analysis and the experimental results of fluorescence power versus excitation power.This letter offers potential value to non-linear optics in biological tissues.     

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.     

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.