2-熒光顯微鏡的基本原理

1、多種研究顯微鏡的使用參考實驗書p155-157倒置顯微鏡及熒光顯微鏡p204 GFP有關(guān)內(nèi)容光學(xué)顯微鏡的基本參數(shù)主要技術(shù)參數(shù)數(shù)值孔徑NA一、倒置顯微鏡(inverted microscope): 物鏡、聚光鏡等位置顛倒；物鏡聚光鏡的工作距離較長，可將盛有樣品的培養(yǎng)皿、培養(yǎng)瓶直接放置在載物臺上進行觀察。二、體視顯微鏡(熒光)三、熒光顯微鏡(fluorescence microscope)： 利用較短波長的光(激發(fā)光)照射樣品，使樣品受到高能量激發(fā)，產(chǎn)生較長波長的熒光(發(fā)射光)，用來觀察和分辨樣品中產(chǎn)生熒光的物質(zhì)的成分和位置。Figure 9-14 Molecular Biology of the

2、 Cell ( Garland Science 2008)The maximum excitation and emission wavelengths of several commonly used fluorescent probes are shown in relation to the corresponding colors of the spectrum.The optical system of a fluorescence microscope.A filter set consists of two barrier filters (1 and 3) and a dich

3、roic mirror (beam-splitting 2)熒光顯微鏡熒光顯微鏡結(jié)構(gòu)及性能特點 熒光顯微鏡的基本構(gòu)造是由普通光學(xué)顯微鏡加上一些附件(如熒光光源 、激發(fā)濾片、雙色束分離器和阻斷濾片等)的基礎(chǔ)上組成的。 熒光光源：高壓汞燈 50200W濾色系統(tǒng) 激發(fā)濾色片：置于光源與物鏡之間，用于選擇激發(fā)光范圍，只有能激發(fā)熒光染料發(fā)光的特定波長的光通過。 阻擋濾色片：物鏡和目鏡之間，選擇性通過特異的熒光，呈現(xiàn)專一的熒光色彩。注意事項：1 暗室內(nèi)進行，打開汞燈5-10min穩(wěn)定后再觀察。2 熒光淬滅(光漂白)：激發(fā)光長時間照射會發(fā)生熒光減弱或消失，操作時注意及時用擋板阻擋激發(fā)光照射標本。3 汞燈關(guān)閉

4、后需冷卻30min后再重新啟動（最好不要短時間內(nèi)反復(fù)開關(guān)，標本應(yīng)集中觀察。）4 載物臺前方黃色遮光板：最好不直接用肉眼看激發(fā)光，以防短波光中的紫外傷害。LEICA TCS- SP2 四、激光掃描共聚焦顯微鏡The confocal fluorescence microscopea mitotic fertilized egg from a sea urchin (Psammechinus) was lysed with a detergent, exposed to an anti-tubulin antibody. Confocal microscopy produces an in-foc

5、us optical section through thick cells.普通熒光顯微鏡下，存在許多來自焦平面以外的熒光，使觀察到得圖像反差和分辨率降低。Confocal激光共聚焦掃描顯微鏡在某一瞬間只用很小的一束光激發(fā)，發(fā)出的熒光通過一個pinhole小孔/狹縫后成像，保證只有來自該焦平面的光成像，來自焦平面以外的雜散光則被pinhole小孔擋住。所得圖形更加清晰。 激光共聚焦顯微鏡的分辨率比普通熒光顯微鏡的分辨率提高1.4-1.7倍。When viewed by conventional fluorescence microscopy, the image is blurred. Th

6、is blurring occurs because background fluorescence is detected from tubulin above and below the focal plane.The confocal microscopic image is sharp, particularly in the center of the mitotic spindle. In this case, fluorescence is detected only from molecules in the focal plane, generating a very thi

7、n optical section.The emitted fluorescent light passes through a pinhole that rejects out-of-focus light, thereby only light in focus with the image is collected.XYZFigure 9-22 Molecular Biology of the Cell ( Garland Science 2008)3-D reconstruction from confocal microscope images.立體三維重組功能Pollen grains, from a passion flower, have a complex sculptured cell wall that