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1、Fundamentals of Light Microscopy,Junle QuE-mail: 深圳大学光电工程学院,Chapter 1,2,Microscope-显微镜,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,
2、Microscope: instrument to see objects too small for the naked eye.,It is difficult to say who invented the compound microscope.,History of invention,http:/en.wikipedia.org/wiki/Microscope,3,Three Goals of Microscopy,M: produce a magnified image of the specimenR: separate the details in the imageC: r
3、ender the details visible to the human eye or camera.,MRC: Magnification, Resolution, Contrast,4,History of Light Microscopy,A simple compound microscope invented by British microscopist Robert Hooke sometime in the 1660s.,5,6,7,History of Light Microscopy,8,Invented by Robert Hooke in1600s,Microsco
4、pe,20th century microscopes,21th century microscopes,9,Microscope & Microscopy & Microscopic,Microscope (显微镜)instrument to see objects too small for the naked eye. Microscopy (显微成像方法)the science of investigating small objects using such an instrument. Microscopic (用显微镜可见的)invisible to the eye unless
5、 aided by a microscope.,10,Microscope Elements,Ocular lens (eyepiece) (1)-目镜 Objective (3) 物镜Diaphragm and condenser lens (8) 光栏和聚光镜Light source, a light or a mirror (7),Basic optical transmission microscope elements,Objective turret (2) （目镜架）Stage (to hold the sample) (6 ) 、 ( 9) 样品台Focus wheel（聚焦轮
6、） to move the stage (4 - coarse adjustment, 5 - fine adjustment),Basic components of the light path：光路部分构成,“Structural” components:结构部分,11,What is an eyepiece (or ocular)目镜?,An eyepiece, or ocular lens: is a type of lens that is attached to a variety of optical devices such as telescopes（望远镜) and mi
7、croscopes(显微镜）.named because it is usually the lens that is closest to the eye,A collection of different types of eyepieces,A 25 mm Kellner eyepiece,12,What is eyepiece (or ocular)?,An eyepiece is usually described by barrel diameter and focal length. Common barrel diameters are 0.96, 1.25 (standard
8、) and 2. Commonly available eyepiece focal lengths range from 2mm to 40mm.,1-Real image 2- Field diaphragm 3- Eye relief 4- Exit pupil,The eyepiece is placed at the focal point of the objective,13,What is objective?,Objective: the optical element that gathers(收集) light from the object being observed
9、 and focuses the light rays to produce a real image(实像）. named because it is usually the lens that is closest to the object,Leica oil immersion microscope objective,14,15,How image becomes magnified in microscope?,16,How image becomes magnified in microscope?,17,How image becomes magnified in micros
10、cope?,18,How image becomes magnified in microscope?,19,How image becomes magnified in microscope?,20,Basic Configuration: Inverted Microscope（倒置显微镜）,inverted microscope for fluorescence microscopy（倒置荧光显微镜）.,Inverted microscope: light source and condenser on the top, above the stage pointing down, wh
11、ile the objectives and turret are below the stage pointing up.,inverted microscope for tissue culture（组织培养用倒置显微镜）.,21,Basic Configuration: Upright Microscope（正置显微镜）,Upright microscope: light source and condenser on the bottom, below the stage pointing up, while the objectives and turret are above th
12、e stage pointing down.,22,Principle of Koeher Illumination: A given 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 照明系统,Koehler illumination, invented in 1893 by August K
13、oehler,How to achieve Koeher Illumination?,23,Koeher(科勒) Illumination System,科勒照明的实现：把光源的发光体正好摆在聚光镜下方的前焦平面,Koeher照明光路,发光体上点光源变成一束平行光（光源上一点照明样品的每一点）整个发光体变成无数束平行光（样品上一点由光源每一点照明）,24,Koeher(科勒) Illumination System,Field planes and Aperture planes,eyepieces,eyepiecetelescope,25,Diaphragm,光阑分为孔径光阑和视场光阑、渐晕
14、光阑和杂消光光阑等,孔径光阑：用于确定辐射测量的面积的光阑（改变成象光束的大小和控制进入光学系统的光通亮）,视场光阑：限制成像范围的光阑,26,Field stop diaphragm (视场光阑),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 an
15、d objective Can Not affect the Resolution,Function of the field stop diaphragm in Microscope,27,Aperture diaphragm (孔径光阑),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 an
16、d focal depth. Can Not affect the viewing field,Function of the aperture diaphragm in Microscope,28,Aperture diaphragm,Function of the aperture diaphragm in Microscope Adjust the NA of the condenser lens,NA=1.2,NA=0.6,NA=0.3,29,Aperture diaphragm,Function of the aperture diaphragm in Microscope Adju
17、st the contrast resolution,聚光镜孔径光阑=NA,聚光镜孔径光阑=0.8NA,聚光镜孔径光阑=0.6NA,聚光镜 孔径光阑的大小缩小到等于所用物镜出射光瞳的2/3或4/5效果好, 这是在最大分辩率与最佳反差间所作的折衷.,30,Adjusting Microscope for Koeher Illumination,1. Preliminaries,specimen slide condenser objective diaphragms lamp power,31,Adjusting Microscope for Koeher Illumination,2. Lam
18、p fills the front aperture of the condenser,Purpose: to achieve Koeher IlluminationTool: eyepiece telescope or Bertrand lens condenser objective diaphragms lamp power,32,Adjusting Microscope for Koeher Illumination,3. Focus the specimen,Purpose: to achieve Koeher IlluminationTool: eyepiece telescope
19、 or Bertrand lens objective focus dials stage controlsNOTED: avoid objective plunge into specimen slide,33,Adjusting Microscope for Koeher Illumination,4. Focus and center the condenser,Purpose: reduce scattered light and improve image contrast condenser field diaphragm lamp power,34,Adjusting Micro
20、scope for Koeher Illumination,5. Adjust the condenser diaphragm,Purpose: to obtain the best resolution and contrastTool: eyepiece telescope or Bertrand lens condenser diaphragmNOTED:3/4 of the maximum diameter of the aperture,35,Adjusting Microscope for Koeher Illumination,6. Adjust the brightness,P
21、urpose: to obtain optimal image brightness voltage neutral density filtersNOTED: aperture diaphragm should never be closed downs as a way to reduce light intensity,36,37,38,39,40,41,42,Detectors for Optical Microscopy,Film photomicrographyVideo imaging (Close Circuit TV)Digital imaging with a charge
22、 coupled device (CCD)Scanning microscopyPhotomultiplier tube (PMT)-光电倍增管Avalanche photodiode (APD)-雪崩光电二极管,43,44,What is Contrast（对比度）?,Differences in light intensity, phase, polarization, color create image contrast, which allows individual features and details of the specimen to become visible. Co
23、ntrast 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%,45,Contrast Mechanisms（都有哪些对比度）,Bright field (low contrast) 明场Phase contrast 相位衬度Differential interference
24、contrast (DIC) 微分干涉Dark field contrast 暗场Polarization microscopy 偏振显微Fluorescence microscopy Chemical SelectivityVibration microscopy 振动显微,46,Summarization,Optical Microscope is a powerful tool in many fields including life science, clinics, material and chemistry.Khler Illumination: standard for de
25、signing an optical microscopeComponents of a microscope:Objective lens (Aberration correction, NA, working distance)Detectors (CCD, PMT)Contrast mechanismsPhase contrastDIC contrastDark field contrastFluorescenceReference:http:/micro.magnet.fsu.edu/primer/virtual/confocal/index.html,47,Quiz,Who inve
26、nted the first microscope?What are the goals of microscopy?What are the main components of an optical microscope?What is the overall magnification of the microscope?Do you have any experience with microscopy? Please describe.How are the microscopes classified in terms of their structures?What is the contrast of optical microscope? Please give a definition and its expression using an equation.What are the common contrast mechanisms for optical microscopy?,
