传统明场成像 d b a 6
传统明场成像 6
景深D:物镜的垂直分辨率 (0.15~0.30)n (N.A.)M (uum) Depth of Field Ranges Low NA High NA Object Plane Depth of In a conventional widefield optical epi- Field fluorescence microscope,secondary fluorescence emitted by the specimen often occurs through the excited volume and obscures resolution of features that lie in the objective focal plane. Figure 1
7 景深Df：物镜的垂直分辨率 In a conventional widefield optical epifluorescence microscope, secondary fluorescence emitted by the specimen often occurs through the excited volume and obscures resolution of features that lie in the objective focal plane
共聚焦成像 C d b a 非焦平面 杂散光 8
共聚焦成像 8 非焦平面 杂散光
激光共聚焦显微镜 Modern confocal microscopes can be considered as completely integrated electronic systems where the optical microscope plays a central role in a configuration that consists of one or more electronic detectors,a computer (for image display,processing,output,and storage),and several laser systems combined with wavelength selection devices and a beam scanning assembly. Photomultiplier Detector Confocal Microscopy Information Flow Schematic Diagram Detector Laser Scanning Pinhole Confocal Microscope Low Noise Frame Aperture Photomultiplier Optical Store Video Tube Out-of-Focus Display Video Fluorescence- Configuration Light Rays Scanning Prints. Computer Barrier Unit Slides Filter Excitation Laser Filter Excitation In-Focus Source Air Cooled Confocal Light Rays Krypton Pinhole Argon Laser Barrier Filter SYNC Dichromatic 3-D Reconstruction Mirror Dichroic Excitation Mirror Light Exciter Objective Rays Filter Light Source Galvanometer Pinhole X,Y Syncro Scanning Digital Hardcopy Aperture Unit Scan and Backup Control Stepper Motor Focal High NA IFocus X-Y Stage for Z-Series Planes Objective Figure 2 Lens Cover Slip Controller Shutter Optical System Control Confocal Pinhole Control Specimen Specimen- Slide ◆Laser Power Photomultiplier◆ Figure 5 Tube Gain Filter Wheels 9
激光共聚焦显微镜 • Modern confocal microscopes can be considered as completely integrated electronic systems where the optical microscope plays a central role in a configuration that consists of one or more electronic detectors, a computer (for image display, processing, output, and storage), and several laser systems combined with wavelength selection devices and a beam scanning assembly. 9
荧光显微镜 The entire specimen is subjected to intense illumination from an incoherent mercury or xenon arc-discharge lamp,and the resulting image of secondary fluorescence emission can be viewed directly in the eyepieces or projected onto the surface of an electronic array detector or traditional film plane. Kohler lllumination in Reflected Light Fluorescence Image Field Plane Diaphragm Controls Field Collector Diameter Field Arc Lamp Lens Lens Plasma Ball -Emission (Barrier) Filter Cover Glass. Dichromatic Specimen- Mirror Microscope Slide Objective Widefield illumination Excitation Rear Focal (Large Volume) Beam Arc Image Excitation Plane Filter at Condenser Aperture Diaphragm Condenser and Objective Figure 5 10 Specimen-
荧光显微镜 • The entire specimen is subjected to intense illumination from an incoherent mercury or xenon arc-discharge lamp, and the resulting image of secondary fluorescence emission can be viewed directly in the eyepieces or projected onto the surface of an electronic array detector or traditional film plane. 10