光學(xué)顯微與電子成像1

1、Fundamentals of Light MicroscopyJunle QuE-mail: jlqu深圳大學(xué)光電工程學(xué)院Chapter 1Microscope-顯微鏡 The oldest published image known to have been made with a microscope (1630)It is often said that Dutch spectacle-makers Hans Janssen and his son Zacharias Janssen to invent the first compound microscope in 1600s Mi

2、croscope: instrument to see objects too small for the naked eye. It is difficult to say who invented the compound microscope. History of invention /wiki/Microscope2Three Goals of MicroscopyM: produce a magnified image of the specimenR: separate the details in the imageC: render the details visible t

3、o the human eye or camera. MRC: Magnification, Resolution, Contrast3History of Light MicroscopyA simple compound microscope invented by British microscopist Robert Hooke sometime in the 1660s. 456History of Light Microscopy7Invented by Robert Hooke in1600s Microscope20th century microscopes 21th cen

4、tury microscopes 8Microscope & Microscopy & MicroscopicMicroscope (顯微鏡)instrument to see objects too small for the naked eye. Microscopy (顯微成像方法)the science of investigating small objects using such an instrument. Microscopic (用顯微鏡可見的)invisible to the eye unless aided by a microscope.9Microscope Ele

5、mentsOcular lens (eyepiece) (1)-目鏡 Objective (3) 物鏡Diaphragm and condenser lens (8) 光欄和聚光鏡Light source, a light or a mirror (7) Basic optical transmission microscope elements Objective turret (2) （目鏡架）Stage (to hold the sample) (6 ) 、 ( 9) 樣品臺(tái)Focus wheel（聚焦輪） to move the stage (4 - coarse adjustment

6、, 5 - fine adjustment) Basic components of the light path：光路部分構(gòu)成“Structural” components:結(jié)構(gòu)部分 10What is an eyepiece (or ocular)目鏡?An eyepiece, or ocular lens: is a type of lens that is attached to a variety of optical devices such as telescopes（望遠(yuǎn)鏡) and microscopes(顯微鏡）.named because it is usually th

7、e lens that is closest to the eye A collection of different types of eyepiecesA 25 mm Kellner eyepiece11What is eyepiece (or ocular)?An eyepiece is usually described by barrel diameter and focal length. Common barrel diameters are 0.96, 1.25 (standard) and 2. Commonly available eyepiece focal length

8、s range from 2mm to 40mm. 1-Real image 2- Field diaphragm 3- Eye relief 4- Exit pupilThe eyepiece is placed at the focal point of the objective 12What is objective?Objective: the optical element that gathers(收集) light from the object being observed and focuses the light rays to produce a real image(

9、實(shí)像）. named because it is usually the lens that is closest to the objectLeica oil immersion microscope objective1314How image becomes magnified in microscope?15How image becomes magnified in microscope?16How image becomes magnified in microscope?17How image becomes magnified in microscope?18How image

10、 becomes magnified in microscope?19Basic Configuration: Inverted Microscope（倒置顯微鏡）inverted microscope for fluorescence microscopy（倒置熒光顯微鏡）. Inverted microscope: light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. inve

11、rted microscope for tissue culture（組織培養(yǎng)用倒置顯微鏡）.20 Basic Configuration: Upright Microscope（正置顯微鏡） Upright microscope: light source and condenser on the bottom, below the stage pointing up, while the objectives and turret are above the stage pointing down. 21 Principle of Koeher Illumination: A given

12、point in the specimen is illuminated by every point in the light sourceA given point in the light source illuminates every point in the specimen. Koeher(科勒) Illumination System 照明系統(tǒng)Koehler illumination, invented in 1893 by August KoehlerHow to achieve Koeher Illumination?22 Koeher(科勒) Illumination S

13、ystem科勒照明的實(shí)現(xiàn)：把光源的發(fā)光體正好擺在聚光鏡下方的前焦平面Koeher照明光路發(fā)光體上點(diǎn)光源變成一束平行光（光源上一點(diǎn)照明樣品的每一點(diǎn)）整個(gè)發(fā)光體變成無數(shù)束平行光（樣品上一點(diǎn)由光源每一點(diǎn)照明）樣品上無燈絲像，無條紋23 Koeher(科勒) Illumination SystemField planes and Aperture planeseyepieceseyepiecetelescope孔徑光闌視場(chǎng)光闌24 Diaphragm光闌分為孔徑光闌和視場(chǎng)光闌、漸暈光闌和雜消光光闌等孔徑光闌：用于確定輻射測(cè)量的面積的光闌（改變成象光束的大小和控制進(jìn)入光學(xué)系統(tǒng)的光通亮）視場(chǎng)光闌：限制成像

14、范圍的光闌25 Field stop diaphragm (視場(chǎng)光闌) Limit the area and the position of illumination on the specimen. Limit the amount of light cast Eliminate flares and ghosting, leading to sharper images. Can Not adjust the NA of the condenser lens and objective Can Not affect the ResolutionFunction of the field s

15、top diaphragm in Microscope 26 Aperture diaphragm (孔徑光闌) Limits the projection of the light source (filament), which enables adjustment of brightness of the field of view.Adjust the angle of incidence. Adjust the contrast resolution and focal depth. Can Not affect the viewing field Function of the a

16、perture diaphragm in Microscope 27 Aperture diaphragm Function of the aperture diaphragm in Microscope Adjust the NA of the condenser lensNA=1.2 NA=0.6 NA=0.3 28 Aperture diaphragm Function of the aperture diaphragm in Microscope Adjust the contrast resolution 聚光鏡孔徑光闌=NA聚光鏡孔徑光闌=0.8NA 聚光鏡孔徑光闌=0.6NA 聚

17、光鏡 孔徑光闌的大小縮小到等于所用物鏡出射光瞳的2/3或4/5效果好, 這是在最大分辯率與最佳反差間所作的折衷.29 Adjusting Microscope for Koeher Illumination1. Preliminaries specimen slide condenser objective diaphragms lamp powerspecimen planeeyepointimage formed by objectivecondenseraperture diaphragmfield stop diaphragm30 Adjusting Microscope for Ko

18、eher Illumination2. Lamp fills the front aperture of the condenserPurpose: to achieve Koeher IlluminationTool: eyepiece telescope or Bertrand lens condenser objective diaphragms lamp power31 Adjusting Microscope for Koeher Illumination3. Focus the specimenPurpose: to achieve Koeher IlluminationTool:

19、 eyepiece telescope or Bertrand lens objective focus dials stage controlsNOTED: avoid objective plunge into specimen slide32 Adjusting Microscope for Koeher Illumination4. Focus and center the condenserPurpose: reduce scattered light and improve image contrast condenser field diaphragm lamp power33

20、Adjusting Microscope for Koeher Illumination5. Adjust the condenser diaphragmPurpose: to obtain the best resolution and contrastTool: eyepiece telescope or Bertrand lens condenser diaphragmNOTED:3/4 of the maximum diameter of the aperture34 Adjusting Microscope for Koeher Illumination6. Adjust the b

21、rightnessPurpose: to obtain optimal image brightness voltage neutral density filtersNOTED: aperture diaphragm should never be closed downs as a way to reduce light intensity35363738394041Detectors for Optical MicroscopyFilm photomicrographyVideo imaging (Close Circuit TV)Digital imaging with a charg

22、e coupled device (CCD)Scanning microscopyPhotomultiplier tube (PMT)-光電倍增管Avalanche photodiode (APD)-雪崩光電二極管4243What is Contrast（對(duì)比度）?Differences in light intensity, phase, polarization, color create image contrast, which allows individual features and details of the specimen to become visible. Contr

23、ast is defined as the difference in light intensity between the object and the adjacent background relative to the overall background intensity.Percent Contrast = (I(s) - I(b) / I(b) x 100%44Contrast Mechanisms（都有哪些對(duì)比度）Bright field (low contrast) 明場(chǎng)Phase contrast 相位襯度Differential interference contrast (DIC) 微分干涉Dark field contrast 暗場(chǎng)Polarization microscopy 偏振顯微Fluorescence microscopy Chemical SelectivityVibration microscopy 振動(dòng)顯微45SummarizationOptical Microscope is a powerful tool in many fields including life science, clinic