激光共聚焦显微镜原理 激光共聚焦显微镜原理和应用.docVIP

激光共聚焦显微镜原理 激光共聚焦显微镜原理和应用 激光共聚焦显微镜原理和应用 共聚焦显微镜的发展历史 1955年，Marvin Minsky利用共焦原理搭建了一台共焦显微镜，用来在体观察大脑的神经元网络。1957年，Marvin Minsky申请了共聚焦显微镜的专利。1970年，第一台单光束共聚焦激光扫描显微镜问世。1985年，多个实验室的多篇报道显示共聚焦显微镜可以消除焦点模糊，得到非常清晰的图像。1987年，BIO-RAD公司推出了第一台商业化的共聚焦显微镜。 共聚焦显微镜最大的优点就是可以只检测一个聚焦平面的信号。样品聚焦平面和检测器（光电倍增管）之前均有一个针孔，针孔的设置可以有效地滤除非聚焦平面的信号，增加显微镜的信噪比。激光扫描显微镜能够逐点和诸行对样品进行扫描，最终根据象素信息形成一个高对比度和高分辨率的图像。通过逐层对样品扫描并把每一层的图像组合成一个整体，激光扫描显微镜能够对样品进行三维分析，非常适合于超厚样品的检测。传统显微镜是一次性照明整个视野中的样品，因此可以用眼睛直接观察或者用CCD获取图像，没有时间延迟；而共聚焦显微镜是逐点成像，无法用眼睛成像，也无法用CCD获取图像，只能用探测器收集每个象素点的信号，再通过软件重构图象，有一定的时间延迟。 How a Confocal Image is Formed Pinhole 1SpecimenPinhole 2 Detector CondenserLensObjectiveLens Wide Microscopy and Confocal Micoscopy Wide Field Confocal FieldWide FieldConfocal Field Confocal Principle Standard Band Pass Filters 630 nm BandPass Filter White Light Source Transmitted Light620 -640 nm Light Standard Long Pass Filters 520 nm Long Pass Filter Light Source 520 nm LightTransmitted Light Standard Short Pass Filters 575 nm Short Pass Filter Light Source Optical Filters Dichroic Filter/Mirror at 45 deg Light SourceTransmitted LightReflected light510 LP dichroic Mirror 生命科学院的激光共聚焦显微镜 Beam Path of Zeiss CLSM 510 METAThe unique scanning module is the core of the LSM 510 META. It contains motorized collimators, scanning mirrors, individually adjustable and positionable pinholes, and highly sensitive detectors including the META detector. All these components are arranged to ensure optimum specimen illumination and efficient collection of reflected or emitted light. A highly efficient optical grating provides an innovative way of separating the fluorescence emissions in the META detector. The grating projects the entire fluorescence spectrum onto the 32 channels of the META detector. Thus, the spectral signature is acquired for each pixel of the scanned image and subsequently can be used for the digital separation into component dyes. To get an 2 D image, the excitation spot has to be moved over the specimen X X/Y Image Y Spec