上海交通大学：《材料组织结构表征》课程教学资源（课件讲义）2019-光学显微镜4-共聚焦扫描显微镜

scanning mi个r0rS detector pin hole 四、共聚焦扫描显微镜 microscope sample

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共聚焦扫描显微镜 ·成像原理 ·构造 aser ·应用 scanning %i个c0f5 detector pinhole microscope Sample

共聚焦扫描显微镜 • 成像原理 • 构造 • 应用 2

共聚焦光学 ·从一个点光源发射的探测光通过透镜聚焦到被观测物体上，如果物体恰在焦 点上，那么反射光通过原透镜应当汇聚回到光源，这就是所谓的共聚焦，简 称共焦 18 22 24 8 B F1G.1. 光源面 样品焦平面 像平面 共轭 3

共聚焦光学 • 从一个点光源发射的探测光通过透镜聚焦到被观测物体上，如果物体恰在焦 点上，那么反射光通过原透镜应当汇聚回到光源，这就是所谓的共聚焦，简 称共焦 3

共聚焦光学 1957年由马文·闵斯基(Marvin Lee Minsky)提出。 ● “共聚焦显微镜是一种利用空间针孔(spatial pinhole)过滤掉非焦平面光线， 从而提高图像对比度并获得试样三维形貌的光学显微技术。”(Pawley JB (editor)(2006).Handbook of Biological Confocal Microscopy(3rd ed.)) The first commercial instruments appeared in 1987. 18 22 20 24 28 B 4 FIG.I. 光源面 样品焦平面 像平面 共轭 4

共聚焦光学 • 1957年由马文·闵斯基（Marvin Lee Minsky）提出。 • “共聚焦显微镜是一种利用空间针孔(spatial pinhole)过滤掉非焦平面光线， 从而提高图像对比度并获得试样三维形貌的光学显微技术。” (Pawley JB (editor) (2006). Handbook of Biological Confocal Microscopy (3rd ed.)) • The first commercial instruments appeared in 1987. 4

荧光显微镜 在传统的荧光显微镜中，来自汞灯或者氙灯的激发光照射视野中的全部样品， 观察者可以用眼睛直接观察，也可以用CCD等设备直接拍摄荧光图像。 Kohler Illumination in Reflected Light Fluorescence Image Field Plane Diaphragm Controls Field Collector Diameter Field Arc Lamp Lens Lens Plasma Ball -Emission (Barrier) Filter Dichromatic -Mirror -Objective Rear Focal Arc Image Excitation Plane Filter at Condenser Aperture Diaphragm Condenser and Objective Figure 5 Specimen- 5

荧光显微镜 • 在传统的荧光显微镜中，来自汞灯或者氙灯的激发光照射视野中的全部样品， 观察者可以用眼睛直接观察，也可以用CCD等设备直接拍摄荧光图像。 5

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 epi￾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

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