光学显微与电子成像-激光共焦显微镜.ppt

CONFOCAL MICROSCOPY *** 2014.11.24 Chapter 12 Outline 1. Principle of confocal microscope Scan modes Image generation Advantages 2. The factors affecting the quality of the image Pinhole size Scan rate Zoom Principles of Confocal microscopy Principles of confocal microscopy Scan modes Image generation Advantages Principles of Confocal microscopy In confocal system: A point light source for illumination A point light focus within the specimen A pinhole at the image detecting plane What is confocal? These three points are optically conjugated together and aligned accurately to each other in the light path of image formation this is confocal. Principles of Confocal microscopy Principles of Confocal Microscopy 3213 PMT, for TPEF PMT ( transmission ) PMT(4 Channels) For fluorescence K-Scanner Z-axis scan device Dichromatic Mirror Cooling device for scanner Pinhole lens Prism UV Pinhole lens Configuration of Confocal microscopes (Leica) Pinhole Principles of Confocal microscopy Principles of confocal microscopy Scan modes Image generation Advantages Scan modes Beam scan system single-beam scanning (currently most popular, used in the LSCM ) multiple-beam scanning Scan modes single-beam scanning(currently most popular, used in the LSCM ) Scan modes Scanning a single mirror on two orthogonal axes two-mirror configuration single-beam scanning 00 Scan modes multiple-beam scanning Scan modes Each disk has more than 20,000 pinholes. Principles of Confocal microscopy Principles of confocal microscopy Scan modes Image generation Advantages Image Generation Original design: A real image was not formed in Minskys original design, but instead the output from the photomultiplier was translated into an image on the screen of a military surplus long persistence oscilloscope that had no provision for recording. Image Generation PMT: detects light and converts photon hits into analogue electron flow. ADC: analogue electron signal is