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：     

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。     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像     

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.     

光学仪器

眼镜、放大镜、显微镜、望远镜TH742.652006021689高斯光束荧光共焦显微镜的三维光学传递函数=Opticaltransfer function of a fluorescent confocal microscope withextended Gaussian source[刊,中]/杨初平(华南农业大学理学院.广东,广州(510642))∥激光技术.—2005,29(5).—552-554研究高斯光源的束斑半径、光源孔径和探测器孔径对荧光共焦显微镜3-D OTF的影响,获得了具有有限光源孔径、探测器孔径和束斑半径的荧光共焦显微镜的三维光学传递函数(3-D OTF)。数值计算结果表明,束斑半径影响到光源孔径和探测器孔径的选择,与采用平行光…     

荧光波长对共焦显微镜成像特性的影响

导出了共焦显微镜中不同荧光波长情况下的荧光功率传输函数、三维脉冲响应函数（3D－PSF）和三维光学传递函数（3D－OTF）。结果表明，不同的荧光波长对共焦显微镜的空间截止频率、分辨率、光学传递函数存在明显的影响。当激发波长与荧光波长的比值下降到一定程度时，可以看到明显的失锥现象。     

内窥式散斑类共聚焦系统层析能力分析

本文研究了内窥式散斑类共聚焦系统(Endoscope-based speckle quasi-confocal system,EBSQCS)的层析能力。从散斑类共聚焦显微镜(Speckle quasi-confocal microscope,SQCM)成像原理出发,详细分析了内窥镜光学结构对散斑类共聚焦显微镜散斑场波动的影响规律,推导了内窥镜光学结构与内窥式散斑类共聚焦系统轴向分辨力的关系。实验测得了基于光纤束的内窥式散斑类共聚焦系统的轴向分辨力曲线。选用放大倍率4倍,光纤直径5μm的内窥镜系统的内窥式散斑类共聚焦系统轴向分辨力曲线的全峰半高是散斑类共聚焦显微镜的2.3倍,与理论计算值相符。实验结果表明内窥式散斑类共聚焦系统具有很好的轴向层析能力。     

细胞研究的光学显微成像

介绍了在细胞及亚细胞结构与功能研究中发挥至关重要作用的各种现代光学显微镜,包括近场显微镜、共焦扫描显微镜、双光子成像显微镜、图像恢复显微镜及时间相关单光子计数技术,着重描述了后两种系统的工作原理及先进功能。     

图像复原式整形环形光横向超分辨共焦显微测量新方法

提出一种新的图像复原式整形环形光横向超分辨共焦显微测量法. 该方法首先利用二元光学器件，将高斯照明光束整形为环形光束，用于初步改善共焦显微镜的横向分辨力，然后利用基于最大似然估计法(maximum likelihood estimate, MLE)的单幅图像超分辨复原技术，重建测量图像的高频信息，来进一步改善共焦显微镜的横向分辨力. 实验表明,当λ=632.8nm，N.A. =0.85时，该方法能使共焦显微镜获得优于0.1μm的横向分辨力. 利用该方法建立的横向超分辨共焦显微系统除了具有显著的超分辨效果外 关键词： 超分辨 超分辨复原 最大似然估计 共焦成像     

线结构光共焦显微成像技术的实验研究

激光共焦扫描显微镜大多采用点共焦扫描成像形式,扫描机构复杂,成本高。新型的线结构光共焦显微成像技术是对样品进行线共焦成像,只需对样品进行一维扫描就可以得到整个平面的像。设计了线结构光共焦成像实验装置,进行了线结构光共焦显微成像实验。实验结果证明了线结构光共焦成像的可行性,杂散光明显地减少,提高了成像清晰度,并且有光学层析能力。采用低照度高分辨率CCD代替价格昂贵的像增强器大大降低了系统成本。     

Some efficient methods to correct confocal images for easy interpretation

In this paper we have explained some efficient methods to correct artefacts in confocal laser beam scanning microscope (CLSM) images. The main aim is to enhance object features such that they become clearly visible for interactive evaluation and to reduce the overall noise so that the automatic segmentation and feature measurement can be done easily. A simple automatic-thresholding technique, and a straightforward method to restore the light intensity along the depth of the image stack are proposed. Another problem associated with the CLSM is the non-isotropic resolution. We have presented an interpolation technique based on XOR contouring and morphing to virtually insert the image slices in the image stack for improving the axial resolution. This interpolation technique has the merits of both contour- and intensity-based interpolations. Results of application of these methods on CLSM data are shown.     

The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.     

Facile method for CLSM imaging unfunctionalized Au nanoparticles through fluorescent channels

The microscopic visualization of metal nanoparticles has become a useful tool for the investigation of their applications in cell labeling and the study of their bio-effects. In the current study, we have developed a facile method with confocal laser scanning microscope (CLSM) to observe unfunctionalized Au nanoparticles through fluorescent channels. The sharp reflected signal and photostable property of the metal nanoparticles makes the present method very ideal for fluorescent co-localization, real-time imaging, and further quantitative analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lan Yuan and Wei Wei contributed equally to this study.     

Sonodynamic effects of protoporphyrin IX disodium salt on Ehrlich ascetic tumor cells

The cytotoxic effect of protoporphyrin IX disodium salt (PPIX) on isolated Ehrlich ascetic tumor (EAT) cells induced by ultrasound exposure was investigated. Tumor cells suspended in air-saturated phosphate buffer solution (PBS, pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presence and absence of PPIX. The viability of cells was determined by a trypan blue exclusion test. The morphological changes of cells in SDT were observed by scanning electron microscope (SEM). And the sub-cellular localization of PPIX in EAT cells was detected by confocal laser scanning microscopy (CLSM). The ultrasonically-induced cell damage increased as PPIX concentration increased, while no cell damage was observed with PPIX alone. CLSM observation revealed that the fluorescence of PPIX and rhodamine 123 (mitochondrial probe) overlapped very well in the cytoplasm. The results indicate that PPIX could enhance the ultrasonically-induced cell damage and mitochondria may play an important role during sonodynamically induced cytotoxicity.     

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5 MHz for 10 min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.     

Effects of laser power density on the microstructure and microhardness of Ni–Al alloyed layer by pulsed laser irradiation

Laser alloying of Ni–P electroless deposited layer with aluminum substrate was carried out by Nd–YAG pulsed laser. The phase composition and microstructure of the alloyed layers produced by different laser power densities were identified by X-ray diffractionary (XRD), scanning electron microscope (SEM) accompanied by energy dispersion X-ray analysis (EDS) and transmission electron microscope (TEM). Furthermore, the surface roughness of the alloyed layers was characterised by confocal laser scanning microscope (CLSM). The results showed that the characteristic dendritic or lamellar microstructures were observed in the alloyed layers. The phase constituents of the alloyed zones were intermetallic compounds of nickel–aluminum NiAl, Al₃Ni and Al₃Ni₂, as well as some non-equilibrium phases and amorphous phases depending on the employed laser power density. As a result, the microhardness of the alloyed layer with Ni–P amorphous phases formed at laser power density 5.36×10⁹ W/m² reached to HV_0.1 390.     

Sequential confocal and multiphoton laser scanning microscopy using a single photonic crystal fibre based light source

A novel approach that simplifies the laser source requirements for confocal and multiphoton laser scanning (CLSM and MPLSM) using the novel dispersion properties of photonic crystal fibre (PCF) is reported. By tuning the fs-pulsed Ti:Sapphire laser to the zero dispersion wavelength of the PCF, the spectral and temporal properties of the source are largely unaffected and hence this source can easily be used for MPLSM. Conversely, by tuning the Ti:Sapphire laser emission wavelength by less than 10 nm to anomalously pump the PCF, the resultant white-light supercontinuum source can perform CLSM. Sequential CLSM and MPLSM of a double-labelled guinea pig detrusor (smooth muscle layer) specimen is described. PACS 87.64.Tt; 87.64.Vv; 42.65.Ky     

Use of confocal laser scanning microscopy (CLSM) for depthwise resolved microscale-particle image velocimetry (μ-PIV)

A novel use of confocal laser scanning microscopy (CLSM) makes the truly focused field-of-view with well-defined depthwise resolution possible for microscale particle image velocimetry (μ-PIV) applications. The operating principle of the CLSM is presented using the point spread function (PSF) that describes diffracted images of extremely small particles. The implemented high-speed CLSM system using a Nipkow rotating disk is applied to measure the microscale rotating Couette flow field confined between two parallel horizontal disks that are 180-μm apart, with the bottom one stationary and the top one rotating and seeded by 200-nm fluorescent spheres. The CLSM provides much distinct particle images in comparison with the conventional wide-field microscopy (WFM) and the measured vector profiles are more concentric and accurate depicting closer to an ideal Couette flow.     

激光扫描实时共聚焦显微成像系统设计

共聚焦扫描显微镜已成为生物医学和材料科学领域研究中非常有价值的一种工具.本文给出了一种反射型激光扫描共聚焦显微成像系统的系统结构和具体设计.采用多面体转镜进行水平扫描,摆镜进行垂直扫描.利用商品透镜设计了光学扫描中继系统,采用光电倍增管作为激发出的荧光探测器,同时给出了数据采集和扫描同步控制系统的组成与设计.利用COD...     

激光扫描实时共聚焦显微成像系统设计

共聚焦扫描显微镜已成为生物医学和材料科学领域研究中非常有价值的一种工具.本文给出了一种反射型激光扫描共聚焦显微成像系统的系统结构和具体设计.采用多面体转镜进行水平扫描,摆镜进行垂直扫描.利用商品透镜设计了光学扫描中继系统,采用光电倍增管作为激发出的荧光探测器,同时给出了数据采集和扫描同步控制系统的组成与设计.利用CODE V优化光学扫描系统以获得尽可能小的扫描光斑尺寸和较大的视场,并综合考虑了采样频率、扫描速度和探测器对整个系统性能的影响,从而给出了该型共聚焦显微成像系统的相互匹配的设计参量.分析结果表明:共聚焦扫描系统设计合理可行|从光学扫描系统到PMT探测单元的各项技术指标得到优化,满足实时探测的要求|该系统具有适应性强,易升级,低成本的技术特点,同时可达到同类商品的技术性能.