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.     

激光扫描共聚焦显微镜中云纹法的实验研究

将激光扫描共聚焦显微镜与实验力学中的云纹法相结合,提出了激光扫描共聚焦显微镜云纹法并对其进行了实验研究.实验中以激光扫描共聚焦显微镜的扫描线作为虚拟参考栅,以1 200 lines/mm的全息光栅作为试件栅,这两组栅线相干涉形成云纹条纹.对激光扫描共聚焦显微镜中云纹的形成原理和出现条件进行了讨论,对实验结果与理论分析进行了比较.结果表明,激光扫描共聚焦显微镜云纹法是真实可行的.由于该方法无需干涉系统且对测量环境无特殊要求,因此有望在待测物体表面微米量级变形观察和测量上得到应用.     

利用激光共聚焦扫描显微镜的绿色荧光蛋白荧光成像

报字利用基因标记技术净绿色荧光蛋白（ＧＦＰ）基因与目标蛋白基因融合,并利用激光共焦扫描显微镜通过探测绿荧光蛋白的荧光来跟踪观测目标蛋白基因的表达。     

基于共聚焦亚像素扫描的高分辨三维成像

激光雷达技术因具有高精度、高分辨率和工作距离远等优点被广泛应用于三维成像。然而,受光学系统衍射极限的限制,激光雷达的空间分辨率随着目标距离的增大而显著降低。为解决上述问题,结合共聚焦照明技术和亚像素扫描技术,提出一种聚焦照明亚像素扫描光子计数激光雷达,并在实验室内进行了10 m成像实验。结果表明,相较于准直照明光束,采用共聚焦照明光束可将系统空间分辨率由5.0 mm提高到0.9 mm,不仅实现了超光学系统衍射极限成像,还有效降低了多重回波的影响,增强了回波强度。     

共焦激光扫描显微镜技术进展

共焦激光扫描显微镜已延伸到光学显微术领域，包括共焦荧光显微镜、共焦扫描光纤显微镜、彩色共焦激光显微镜、共焦光栅光学显微镜等。并很快成为半导体材料检测和生物医学诊断的重要工具。本文对国外该显微镜技术的特性和新的进展进行了综述。     

共焦激光扫描荧光显微镜的扫描系统研究

本文简述共焦激光扫描灾光显微镜的基本原理，提出扫描系统的方案确定和优化设计的方法，使用该方法何使扫描渐晕减少为原来的１％。同时还给了扫描控制系统运行和控制法以及是均匀图的补偿方法。     

非相干光对共聚焦显微镜层析能力的影响

在共聚焦显微镜中,通过系统的归一化轴向光强半高宽与探测器有效半径和非相干光源有效半径的关系曲线,获得非相干光源照明下系统光学层析能力的变化规律.发现当探测器和光源的有效半径都小于3时,系统具有较好的光学层析能力;其他情况下,系统光学层析能力下降且会受入射光场分布的影响.     

共焦激光扫描荧光显微镜的扫描系统研究

简述共焦激光扫描荧光显微镜的基本原理，给出了扫描系统的方案确定和优化设计的方法，使用该方法可使扫描渐晕减少为原来的１％。同时还给出了扫描控制系统的运行和控制法以及获得均匀图像的补偿方法。     

激光共聚焦扫描显微镜对激光打印文件墨粉厚度的分析研究

使用激光共聚焦扫描显微镜,对六种品牌和型号的激光打印机分别在打印纸和高光相纸上打印的图文墨粉厚度进行测量。结果表明,测量普通打印纸张上激光打印形成墨粉厚度时,测量数据因纸张纤维凹凸不平的影响有一定波动,相纸上激光打印形成的墨粉厚度测量数据相对稳定,并总结出随着打印文件图文线条宽度与墨粉厚度的变化规律。实验表明激光打印文件墨粉厚度的数据测量具有可行性,同一台激光打印机的打印文件墨粉厚度相对稳定,该指标作为一种重要参数可以为激光打印文件检验提供量化依据,并为打印机的同一认定提供依据。     

A Study on Uptake and Localization of Merocyanine 540 (MC540) in Murine Myeloid Leukemia Cells by Confocal Laser Scanning Microscopy (CLSM)

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A Study on Uptake and Localization of Merocyanine 540 (MC540) in Murine Myeloid Leukemia Cells by Confocal Laser Scanning Microscopy (CLSM)

The intracellular localization of merocyanine 540 (MC540), a photosensitizer commonly used in the photo-inactivation of leukemia cells, was studied using confocal laser scanning microscopy. It was found for the first time that MC540 not only localized in the plasma membrane but also in the cytoplasm and the nuclear membrane of the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Exposure of MC540 treated leukemia cells to light under conditions that could cause photobleaching did not cause the redistribution of cell-bound MC540. Rapid localization of MC540 in the cytoplasm was observed 5 minutes after exposure of leukemia cell to MC540, indicating that MC540 could promptly be internalized by these two leukemia cell lines. In contrast, localization of MC540 was limited only to the plasma membrane of erythrocytes. These results suggest that the binding pattern of MC540 is cell type dependent and may be related to the efficacy of photosenitization in photodynamic therapy.     

FTIR及CLSM对转基因杨木细胞壁木质素含量及微区分布研究

通过木质素基因工程能够有效降低杨木细胞壁木质素含量,从而改善人工林杨树作为木质纤维材料的利用现状。选取C3H基因活性抑制表达的转基因杨树和其对照组杨树为实验材料,利用傅里叶红外光谱(FTIR)技术快速表征C3H基因表达活性降低后幼龄杨木细胞壁木质素的含量,并结合激光共聚焦显微镜(CLSM)和组织化学染色技术原位表征木质素含量微区分布变化规律。结果表明转基因杨树与对照组杨树红外谱图的形状和特征峰数目、位置基本一致,表明C3H基因活性降低并未改变杨木细胞壁主要化学成分及结构,但I_{1 508}/I_{1 379},I_{1 508}/I_{1 425}和I_{1 508}/I_{1 740}木质素特征峰高度比值结果表明转基因杨木木质素含量下降了8.2%～9.5%,峰强度的区别说明C3H基因活性抑制表达能够改变杨木细胞壁上木质素等化学组分含量;CLSM观察发现转基因杨木木质素微区分布含量均为纤维细胞角隅>复合胞间层>次生壁,与对照组木质素呈现相同的沉积规律,且转基因杨木细胞壁木质素浓度低于对照组杨木;组织化学染色的结果同样表明杨木S单体木质素均匀分布于纤维细胞壁上,而G单体木质素微区沉积规律为纤维细胞角隅>复合胞间层>次生壁,进一步揭示了C3H基因活性的降低并没有改变转基因杨木G和S木质素单体的沉积规律,但对其纤维和导管壁上木质素单体含量分布有一定影响。     

Three dimensional microvascular measurements in human endometrium using optical slices from laser scanning confocal microscopy (LSCM)

There is increasing interest in the structure of the microvascular environment in human endometrium because of the recognition of the complexity and functional importance of this tissue. Endometrial microcirculatory networks and their relationships have rarely been studied in three-dimensions.Longitudinal uterine slices containing endometrial tissue were carefully selected from women undergoing a hysterectomy. Formalin-fixed endometrial sections (≤50 μm) representing the fundal and isthmic regions were immunofluorescently labeled with monoclonal antibody (CD34) to target the endothelium of microvessel and fluorescein isothiocyanate (FITC) labeled goat anti-mouse. Digital images were acquired using a Nikon Eclipse E800 microscope equipped with a Radiance 2000 confocal scanning laser attachment. ImarisBasic 4.1 visualization suite was utilized for qualitative interpretation. NeuronTracer 1.0 software was utilized to derive the length and numerical densities.There were significant changes across the phases of the menstrual cycle in functional and basal endometrial layers in vessel length density (LD_v) and branch point density (ND_v) within both fundal and isthmic regions of the uterus (P < 0.001). There was also a significant effect of menstrual cycle phase on mean vessel segment length (SL_v) within each region and within each of the layers (P < 0.001). The capillary radial diffusion distance r(diff) was negatively correlated with LD_v. In general, within each of the menstrual cycle phases, LD_v, ND_v were greater in the fundal than the isthmic regions while, in contrast, SL_v was found to be greatest in the isthmic region.Utilization of immunofluorescence and laser scanning confocal microscopy has enabled us to demonstrate significant vascular changes in human endometrial layers illustrating that in general, within each of the menstrual cycle phases, vessel length and branch point densities were greater in the fundal than the isthmic regions, while vessel segment lengths were found to be greatest in the isthmic region.     

Use of Immunofluorescence and Confocal Laser Scanning Microscopy in Identifying Rare Cases of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Showing Dual Specificities to Myeloperoxidase and Proteinase3

Anti-neutrophil cytoplasmic antibodies (ANCA) are the immunodiagnostic markers for idiopathic necrotizing crescentic glomerulonephritis affecting mainly medium to small sized blood vessels. The diagnosis of ANCA associated vasculitis (AAV) is mainly based on clinical and histopathological characteristics along with the serological evidence. Immunofluorescence microscopy (IIF) is considered as the "gold standard" for ANCA detection, and ANCA showing two major patterns ie, cytoplasmic (c-ANCA) and perinuclear (p-ANCA) react with different antigenic targets of neutrophils like Proteinase3 (PR3) and Myeloperoxidase (MPO). A third unusual and rare immunofluorescence pattern called as "X- ANCA" or atypical ANCA is also sometimes seen. The difficulty in identification of ANCA immunofluorescence patterns is mainly seen due to the rare dual patterns seen in the same sera and also the additional nuclear immunofluorescence seen due to presence of anti-nuclear antibodies. ANCA testing by immunofluorescence and Confocal Laser scanning microscopy, as well as by specific ELISAs for detection of anti-PR3 and anti-MPO antibodies have helped in improving the diagnosis. Patients having dual specificities to MPO and PR3 in a patient is a rare finding. Among 425 clinically and histopathologically proven cases of AAV, eight patients (1.9%) had dual specificities, of which five patients showed mixed immunofluorescence pattern and 3 patients showed X-ANCA pattern which was confirmed by both immunofluorescence and Confocal Laser scanning microscopy and the dual specificities to MPO and PR3 were detected by individual ELISAs.     

激光多模式毛细管电泳检测器的光学设计及优化

提出一种基于毛细管电泳的激光多模式检测器.为设计并优化其光路构型,进行了理论分析和数学推导,并采用Matlab仿真,得到光路中各参量优化取值范围.在热透镜通道,激发光束腰半径越小,则热透镜效应越强.而探测激光束腰半径、束腰与样品距离、样品与探测面距离三个参量综合决定检出信号强度;在回射干涉通道,聚焦透镜焦距应较短,它与毛细管距离对检测影响很大,而与激光器、与探测面的两距离对检测影响相对较小;热透镜效应对回射干涉检测影响不明显,如要严格消除,可将两通道错开一适当距离.     

光学相干层析成像系统中双通快速扫描光学延迟线的数值分析

以matlab为工具,详尽分析双通快速扫描光学延迟线在最佳耦合条件下的光束传输与分布,为双通快速扫描光学延迟线的设计、元件选择和系统调整提供理论依据.得出离焦距离Δf=1μm和Δf=578 μm两个理论上的最佳耦合点.但就实际光路调试来看,离焦距离Δf=1 μm为此系统的最佳耦合点.     

激光轰击过程中纳米碳管复合光限幅材料组成及结构的演变研究

光限幅材料在激光轰击过程中的稳定性将在很大程度上决定其实用化价值。文章采用红外(IR)光谱、拉曼(Raman)光谱、透射电子显微镜(TEM)及孔结构分析等测试方法对纳米碳管(CNTs)复合光限幅材料在激光轰击过程中组成、结构的演变进行跟踪研究。结果表明,在强激光轰击下,复合体系中二氧化硅(SiO₂)基质的组成未发生显著改变且网络结构趋于完整,具有较好的稳定性。掺杂CNTs石墨化程度提高,SiO₂凝胶玻璃基质对其起一定的保护作用。轰击过程产生的热效应使得SiO₂颗粒长大,由其堆积而成的孔随之增大。     

可调谐半导体激光吸收光谱技术的调制参量影响及优化选择

为获得较好的谐波信号,完成不同测量目的,以DFB-760.77nm波长处氧气监测为例,通过改变扫描锯齿信号和调制正弦信号的频率和幅值,讨论了调制参量对二次谐波信号峰-峰值、对称性、谐波信号展宽和信号完整性等判别依据的影响和变化.结果表明:Sinmulink模拟值和实验值相关性较高,当正弦调制信号和锯齿扫描信号频率比值为1 000倍时,可以获得较好的二次谐波信号.该研究为实验系统中调制参量的选取和调制度及系统准确度、稳定性、重复性等性能的优化提供了一定帮助.