激光扫描共聚焦显微镜

激光扫描共聚焦显微镜吴旭2008.10.14高级显微镜原理正置、倒置显微镜细胞遗传工作站活细胞工作站激光显微分离系统激光共聚焦显微镜概述激光扫描共聚焦显微镜(Laser scanning coiifocalimcioscope , LSCM )生物医学领域的主要应用通过一种或者多种荧光探针标记后，可对固定的组织或活体样本进行亚细胞水 平结构功能研究高空间分辨率、非介入无损伤连续光学切片、三维图像、实时动态等细胞结构 和功能的分析检测.Conventional fluorescence nucroscope Confocalnucioscope历史1957年，MarvinMmsky提出了共聚焦显微镜技术的某些基本原理，获得了美 国的专利。1967年，Egger和Petian成功地应用共聚焦显微镜产生了一个光学横断面.1977年Sheppard和Wilson首次描述了光与被照明物体的原子之间的非线性 关系和激光扫描器的拉曼光谱学。1984年，Bioiad为公司推出了世界第一台商品化的共聚焦显微镜，型号为 SOM-100.扫描方式为台阶式扫描。1986年MRC-500型改进为光束扫描，用作生 物荧光显微镜的共聚焦系统。Confocal nucioscopy comes of ageJG White & WB Ainos. Nanue 328, 183 -184 (09 July 1987Zeiss、Leica、Mendian、OlympusZeiss LSM510激光扫描共聚焦显微镜Zeiss LSM510 META激光扫描共聚焦显微镜Zeiss LSM510 META激光扫描共聚焦显微镜Nikon AIR激光扫描共聚焦显微镜Prauie UltimalV活体双光子显微镜国家光电实验室（武汉）自制随机定位双光子显微镜Leica TCS SP5激光共聚焦扫描显微镜基本原理相差、DIC常用荧光标记共聚焦原理Two ways to obtain contrast in light microscopy. The stained portions of the cell in(A reduce the amplitude of light waves of paiticulai wavelengths passing thiough them.A colored unage of the cell is thereby obtained that is visible in the oidinaiy way. Light passing tluough the unstained, living cell (B undergoes veiy little change in amplitude, and the stmctuial details cannot be seen even if the image is highly magnified. The phase of the light, however, is altered by its passage tluough the cell, and small phase differences can be made visible by exploiting inteifeience effects using a phase- contiast 01 a diffeiential-inteifeience-contiast micioscope.D. Phase-contiast 01 adifferential-inteiference-contrast microscopeFour types of light nucioscopy. (A The image of a fibroblast in culture obtained by the snnple transmission of light tluough the cell, atechmque known as bnght-field micfoscopy.Tlie other images were obtamed by tecluiiques discussed m the text: (B phase-contrast nucioscopy, (C Nomarski diffeiential-inteifeience-contiast microscopy, and (D daik-field nucioscopy.常用荧光探针Pioteins Nucleic Acids DNA Ions pH Sensitive Indicators Oxidation States SpecificOrganelles荧光显微镜原理明场：透射荧光：落射落射的优点：物镜的聚光镜作用使视场均匀，发射光强度高。激发光损失小，荧光效应高。共聚焦原理由于piiiliole的存在，使得部分杂散光（虚线部分）没有被PMT探测器探测 到，从而提高了成像效果。通过对样品在x-y方向上的逐点扫描，可以形成二维 图像。如果调解焦平面在z方向的位置，连续扫描多个不同z位置的二维图像.则 可以形成一个3D图像，3D的重建需要软件的支持。The unique scaiinmg module is thecoie of the LSM 510 META.It contains motorized collimatois, scaiming muioisandividually adjustable and positionablepnilioles, and highly sensitive detectors mcluding the META detector.All these components areananged to ensure optimum specnnen illumination and efficient collection of reflected 01 emitted light. A highly efficient optical grating provides an iimovative way of separating the fluoiescence emissions in the META detector.The gratmg projects the entire fluoiescence spectnun onto the 32 channels of the META detector. Thus, the spectial signature is acquued fbf each pixel of the scanned unage and subsequently can be used fbf thedigital separation mto component dyes.分光镜组件结构共聚焦构成光学显微镜 激光光源扫描模块（包括共聚焦光路通道和针孔、扫描镜）光检测器计算机系统（数据采集、处理、转换应用）图形输出设备LCSM照片，蓝色为细胞核，绿色为微管图像相关概念RGB三基色图像数据位亮度对比度饱和分辩率怎样得到样品图像样品的标记物有合适的激发波长和发射波长荧光下观察选择合适的图象设置激光扫描的通道参数设置图象的属性调节每一个通道的亮度、对比度、清晰度、背景亮度启动共聚焦和CCD照片的比较更大的动态范围高灵敏度图像背景的消除灵活的拍照区域选择ZOOM功能伪彩色环境要求高多通道图象合成时间序列Ca离子成像FRAP : Fluorescence Redistnbution After Photo bleachingFRAP是用来测定活细胞的动力学参数，借助于高强度脉冲激光来照射细胞某 区域，造成该区域荧光分子的光粹灭，该区域周围的非粹灭荧光分子会以一定的 速率向受照射区域扩散.这个扩散速率可通过低强度激光扫描探测，因而可得到活 细胞的动力学参数。LSCM可以控制光粹灭作用，实时监测分子扩散率和恢复速 率反映细胞结构和活动机制。广泛用于研究细胞骨架构成，核膜结构跨膜大分子 迁移率，细胞间通讯等领域。荧光漂白恢复（FRAP）人卵细胞的局部光漂白FRAP研究ER-GFP的核转位