激光共聚焦显微镜操作方法-confocol
OlympusFV1200激光共聚焦显微镜系统操作手册
Olympus FV1200激光共聚焦显微镜系统操作手册一、开关机步骤1、开机步骤1）打开计算机2）打开汞灯电源开关3）打开显微镜开关4）打开载物台控制器开关5）打开显微镜触屏面板开关6）打开扫描头开关（先开开关键，然后将钥匙顺时针拧到on位置）7）打开激光器①405nm，633nm：打开开关键即可②559nm：先开开关键，绿色指示灯闪烁（约两分钟），停止闪烁后将钥匙顺时针拧到on位置（红灯闪烁，停止闪烁后即开启完成）③多线氩离子（458nm，488nm，514nm）：先开开关键，然后将钥匙顺时针拧到on位置8）双击电脑桌面上打开 FV10-ASW 4.0应用软件。
2、关机步骤1）关闭FV10-ASW 4.0应用软件2）关闭激光器（注意: 多线氩离子458nm，488nm，514nm需先关钥匙，等风机散热20分钟后再关闭开关键）3）关闭扫描头（先将钥匙拧到off位置，再关开关键）4）关闭显微镜触屏面板5）关闭载物台控制器6）关显微镜开关7）关汞灯，先关前面off按钮，倒计时30s，再关后方开关键8）关计算机二、显微镜观察1、用触屏面板选择物镜；2、点击触屏面板EPI，选择需要的荧光滤片，如下图：紫外激发/蓝色光蓝色激发/绿色荧光绿色激发/红色荧光）打开，显微镜目镜下观察样品，推动载物台控制手柄可水平方向移动样品，转动调焦旋钮可调节焦距；三、获图XY多通道扫描1、镜下调好之后，点击软件中的按钮，关闭汞灯快门。
2、首先进行软件设置：在Acquisition Setting里将扫描速度设置为8us/Pixel，像素数一般设置为1024*1024。
在Image Acquisition Control里点击Dye List按钮，在染料列表中，双击用于观察的荧光染料（注：再次双击已选项目可取消），然后点击Apply按钮。
3、将Image Acquisition Control面板中Filter Mode里Kalman 打钩选中，设置线平均Line 2次，这样可以降低图像的噪声。
激光共聚焦显微镜操作规程
激光共聚焦显微镜操作规程一、准备工作a)打开计算机。
依次打开激光器电源、钥匙开关。
多线氩离子(458 nm, 488 nm, 514 nm) ON、氦氖绿(543 nm) ON、氦氖红(633 nm) ON。
打开汞灯电源开关。
b)登陆Windows XP系统。
双击快捷方式：FV10-ASW 1.1。
User ID: Administrator ; Passeword: Administrator。
注意区分大小写。
二、显微镜镜下观察１.微分干涉差观察a)使用手控面板选择物镜。
插入起偏镜。
插入微分干涉滑块。
b)点击FV10-ASW软件中的图标。
标本聚焦.２.荧光观察：c)使用手控面板选择物镜。
d)打开汞灯的机械快门，拉出DIC滑块，点击FV10-ASW软件中的图标e)使用手控面板选择荧光滤色片。
标本聚焦。
３.获取单张荧光图像a)点击FV10-ASW软件中的按钮。
b)关闭汞灯快门，点击按钮，关闭卤素灯快门。
c)点击染料选择按钮，在染料列表中，双击用于观察的荧光染料。
点击Apply按钮。
d)点击XY Repeat按钮开始扫描。
调节图像。
点击Stop按钮停止扫描。
e)选择AutoHV,，并选择扫描速度。
f)点击XY按钮取得一幅图像。
g)点击SeriesDone按钮，“2D View-LiveImage(x)”2D界面就出现。
h)保存该幅图像:右图像管理器中显示的图像图标，选择另存为保存该幅图像。
(保存为“xml”类型是击FV10-ASW软件专用的图像格式。
)４.获得单张（荧光+微分干涉）图像a)点击FV10-ASW软件中的按钮，关闭汞灯快门。
点击按钮，关闭卤素灯快门。
b)点击染料选择按钮，在染料列表中，双击用于观察的荧光染料。
点击Apply按钮。
c)选择TD1。
d)点击XY Repeat按钮开始扫描。
调节绿色(FITC)图像和微分干涉差的图像。
点击Stop按钮停止扫描。
e)选择AutoHV, 并选择扫描速度。
共聚焦使用指南
共聚焦使用指南一、开机1.打开显微镜开关，汞灯开关，电脑开关（地上）。
2.打开激光器开关按钮（右边3个，左边1个（观察DAPI））。
若只分析图片，只开PC/Stand的开关。
3.电脑上打开软件Leica confocal software:公司模式。
4.打开激光外器钥匙（右边3个，左边1个（观察DAPI））。
若只分析图片，不用打开钥匙。
5.488激光调节器（LevelAr/ArKr）至水平状态。
若只分析图片，不用调节此处。
二、显微镜下观察1.放好样品。
2.显微镜身左边：分光拉杆拉至中间，转盘调至相应激发光。
3.显微镜身正前方：Poris调至VIS;MAG调至1x。
4.选择合适倍数调好焦距：荧光弱需要大倍数200x,400x采集图片。
三、电脑下观察采集图片（选择第一通道）1.显微镜转盘转至SCAN，分光拉杆全部推进。
2.显微镜身正前方Poris调至side;MAG调至B灯亮后回调至UV。
3.电脑软件左下角microcontrol选择SCAN。
4.点击软件左下角Beam，选择荧光通道（用途）。
5.双击选择Beam右上角DAPI ，点击软件左下角Continous实时观察。
6.显示窗口Experiment的Qlut看曝光是否过度（蓝色表示过度），可调节Beam左上角的ND值，桌上PMT1和pinhole(此值一般AE在1-1.5，慎调)控制曝光；ZOPS可调节图片Z轴聚焦。
7.点击Beam上Save(回车2次)。
8.Continous调至STOP关闭实时观察。
（选择第二通道）9.双击选择Beam右上角ZZ-FITC，点击软件左下角Continous实时观察。
10.显示窗口Experiment的Qlut看曝光是否过度（蓝色表示过度），可调节Beam上的488通道的百分值，桌上PMT1和pinhole(此值一般AE在1-1.5，慎调)控制曝光；ZOPS可调节图片Z轴聚焦。
11.点击Beam上Save(回车2次)12.Continous调至STOP关闭实时观察（选择第三通道）13.双击选择Beam右上角ZZ-TRITCwide，点击软件左下角Continous实时观察。
激光共聚焦操作步骤
激光共聚焦操作步骤激光共聚焦显微镜（Laser Scanning Confocal Microscope，简称LSCM），是一种光学显微镜，利用激光和扫描镜技术，能够对样品进行高分辨、高对比度的三维成像。
下面将介绍激光共聚焦显微镜的操作步骤。
1.准备工作：a.打开显微镜电源，确保仪器处于正常工作状态。
b.打开激光源电源，等待激光系统启动。
c.准备样品，将样品放置在载玻片上，并使用封口膜或密封胶固定。
2.调节孔径光阑：a.打开镜头盖，取下玻璃保护盖。
b.观察显微镜视野，调节孔径光阑的大小，以获得最佳的入射光。
c.用合适的滤光片选择适当激光激发波长。
3.调节激光功率：a.打开激光源的控制面板。
b.选择合适的激光功率，通常初次使用时可选择较低功率。
c.通过目镜观察激光处于合适的位置和方向，避免直接观察激光。
4.调节焦距：a.打开目镜盖，观察样品上的图像。
b.调节目镜的焦距，获得清晰的图像。
c.使用恰当的倍率进行观察，确保所需的细节可见。
5.调节光路：a.打开激光共聚焦显微镜软件，进行光路调节。
b.调节反射镜和聚焦镜，确保激光能够准确地聚焦在样品上。
c.通过调节扫描镜，使激光能够扫描整个样品。
6.设置扫描参数：a.在软件中选择扫描参数，如扫描速度、像素大小等。
b.减至最小的扫描区域，以便首先观察最感兴趣的区域。
c.如有需要，可以进行连续扫描或者多通道扫描的设置。
7.开始扫描：a.点击软件界面上的“开始”按钮，开始扫描。
b.观察扫描时光点在样品上的移动情况，确保图像的获取和处理是正确的。
c.根据需要调整扫描参数，以获得更好的图像质量。
8.界定感兴趣区域（ROI）：a.选择感兴趣的图像区域，用鼠标进行界定。
b.设置参数，如增益、对比度、亮度等，以便获得更好的视觉效果。
c.按下截图按钮，将感兴趣的图像保存下来。
9.分析和处理图像：a.关闭扫描功能，进入图像处理模式。
b.使用软件提供的功能分析和处理图像，如三维重建、图像拼接等。
Leica激光共聚焦显微镜 操作规程
潍坊医学院医学研究实验中心仪器简明操作规程汇编
激光共聚焦显微镜简明操作规程
1. 启动
开启UPS电源，依次开启右侧手柄上的三个开关，从左至右顺序开启，最后开启激光钥匙。
2. 开启软件
电脑进入操作系统界面后，双击电脑桌面启动共聚焦操作软件。
3. 自检
点击对话框的“OK”键进行自检，结束后进入软件界面
4. 开启激光管
点击屏幕上方的“configuration”按钮进入配置界面→点击左边
按钮→打开所需激光（OFF →ON ），Argon激光还需拖动右方滑块以调节输出功率
5. 设置参数
6. 放置样品进行观察。
7. 观察结束保持数据。
8.关机，做好记录。
激光共聚焦扫描显微镜操作规程
激光共聚焦扫描显微镜操作规程一．开机程序1．开启总电源，确认所有仪器电源状态正常工作2．开启电脑，双击桌面上的LSM510图标，开启显微镜。
3．在初始平面上点击Start Expert Mode 键开启专家拍摄模式。
4．在主菜单中点击File New键，建立新的数据库。
二．软件拍摄1．在主菜单上点击Acquire Laser键，开启需要使用的激光2．在主菜单上点击VIS键，开启显微镜观察模式3．把需要观察的玻片放到载物台上选择需要观察的平面4．在主菜单上点击LSM键，开启进入显微镜激光扫描模式5．在主菜单上点击Config键，选择所需要扫描的模式及参数6．在主菜单上点击Scan Find键，进行图像参数自动校正7．在Scan子菜单下点击Fast X键，进行层面选择8．在连续扫描中调节放大器增益、补偿；激光强度等图像参数来优化图像9．点击图像子菜单上的Save键存储优化好的图像三．关机程序1．完成所有操作后，关掉激光开关，冷却5分钟后，点击主菜单上Exit键，退出所有程序2．关闭电脑3．关闭电源ZEISS 510 MET A二维扫描程序1.在Acquire → config →确定扫描所需的波长，（如果已作过相同方法的扫描，可直接调出一张相同条件的图像，用图像窗中的Reuse确定有关的扫描条件）2.在Acquire → micro →在低倍镜(透射光或荧光)下找到要观察的图像3.在Scan control → mode → find →在显示器上找到图像，选定扫描需用的分辨率4.调节物镜到适当的放大倍数5.Scan control → mode →New，打开一新图像窗6.在Scan control → mode → cont →用zoom将图像大小调至最佳，用Pinhole（一般选1）、Detector Gain（650左右，不宜时间超过800，也不宜低于500）、Amplifier offset（不宜过低），以及选择作算术平均值次数、调节激光强度（488nm不超过25%）等调节图像质量至最佳。
激光扫描共聚焦显微镜操作流程详解
激光扫描共聚焦显微镜操作流程详解激光扫描共聚焦显微镜(laser scanning confocal microscope)激光扫描共聚焦显微镜(laser scanning confocal microscope)，英⽂简写LSCM，俗称confocal。
LSCM是⼀种⾼科技显微镜，属于最先进的细胞⽣物学分析仪器。
我们学院使⽤的是瑞⼠徕卡公司(Leica)的TCS SP5型号的LSCM。
使⽤流程1、登陆ftp://10.31.60.139 或5号楼606室公⽤电脑下载《激光共聚焦预约使⽤审核表》，打印并填写。
2、联系卢剑清（⼿机：135********）审核并签名。
3、持已签名的《激光共聚焦预约使⽤审核表》⾄5号楼610室预约使⽤时间，及领取钥匙，联系⼈：窦凯飞（⼿机：152********）。
4、在熟练使⽤者的指导下，进⾏实验。
5、实验结束，及时归还钥匙。
仪器构造和原理LSCM的基本结构主要包括荧光显微镜系统及样品台、激光发射器、扫描器、检测器、图像存储处理和输出设备、计算机控制系统。
激光光源：激光扫描束经照明针孔形成点光源, 普通显微镜采⽤的⾃然光或灯光是⼀种场光源, 标本上每⼀点的图像都会受到邻近点的衍射光或散射光的⼲扰。
⽽LSCM 以激光为光源, 激光具有单⾊性强﹑⽅向性好﹑⾼亮度﹑相⼲性好等优点, 可以避免普通显微镜的缺点。
⼀般常⽤的⽓体激光器如氩(Ar) ﹑氪(Kr) ﹑氦(He)﹑氖(Ne)。
illuminating pinhole（照明针孔）：使激光经过照明针孔后形成点光源，点光源具有光源⽅向性强、发散⼩、亮度⾼、⾼度的空间和时间相⼲性以及平⾯偏振激发等独特的优点。
且与detector pinhole(探测器针孔)及焦平⾯形成共聚焦装置。
分光镜(BeamSplitter)：点光源发出的光经分光镜反射后, 通过物镜在样品聚焦。
对标本内焦平⾯上的每⼀点进⾏扫描,Focal plane（焦平⾯）：激光点光源照射物体在焦平⾯处聚焦，激发荧光标记的样本发射荧光，形成焦点光斑。
confocal instructions激光共聚焦显微镜教程
University of GuelphCollege of Biological ScienceConfocal Laser Scanning Microscope Instruction(Version 2; 2004.2)ContentsLeica TCS SP2 (2)Sign-up procedure for CLSM on WebCT............................................. (3)Common questions (4)5 Startup......................................................................................... ......... Shutdown. (6)Leica DMR Microscope: general information & care (7)Microscope settingOutward & Koehler illumination setting (9)I: bright field, epifluorescence & laser (all lenses) (10)II: DIC, epifluorescence & laser (63x & 100x lenses) (11)III: Phase contrast, epifluorescence & laser (40x lens) (12)Leica Confocal Software operationAcquisition Dialog Window (13)(Tables for Lens, Format, and Zoom…………...…………… 14)3D Image (18)(Switching z-scan mode; Galvo and Z-wide………………... 18)(When specimens are low signal or bleach sensitive……. 19)Datahandling (20)File types (20)I. Experiment overview window (20)II. Saving Images (21)III. Saving an image from viewer as a hard copy (21)IV. Saving an image as an animation file (21)University of GuelphCollege of Biological Science Confocal Laser Scanning Microscope- Leica TCS SP2 -1. Filter free SP head: a spectrophotometer for each detector channel: design your own filters, maximizesensitivity, minimize crosstalk, record emission spectra.2. Microscope: Upright microscope, Leica DMRo Lenses: 10x, 20x, 40x (Oil, Ph), 63x (Oil, DIC), 100x (Oil, DIC)3. Laser system:o Ar (50 mW): 458 nm, 476 nm, 488 nm, 514 nmo GreenHeNe (1.2 mW): 543 nmo RedHeNe (10 mW): 633 nm4. Two detection channels for fluorescence5. Transmitted light detection6. Leica confocal software (Version 2.5.1227a) for 2 D and 3 D image, ROI scanning, and time courseimaging.7. Simulator for processing images:o Leica confocal softwareo Adobe Photoshop Version 6.0o Office 20008. CD Writer9. Networked in Cbssrv workgroup.Contact: Dr. Michaela Strueder-KypkeOffice: room 208A, Axelrod Bldg.Tel: (519) 824-4120 ext. 52737Fax: (519) 767-1991(c/o the Botany Department)E-mail: confocal@uoguelph.caSign-up Procedure for CLSM on WebCT1. Go to “http://courselink.uoguelph.ca”2. Click on WebCT “Login” button and enter Name and Password inthe appropriate space.3. Under “Courses” heading myWebCT page, click on “LeicaConfocal Laser Scanning Microscope”.4. On the home page, click on “Calendar”.5. Select the month you want to use the microscope.6. Click the day you want to use the microscope.7. You will see the schedule for that day including other sign upsand times. If you click “View Week”, you will see the schedule forthe week.8. If you would like to book a time, go to “Add entry”9. Type your name in “Summary” box. Select your Start and Endtimes in the bottom columns. Keep access level “Public”.- Add in the “Detail” box which lasers you intend to use (Ar,GreenHeNe, HeNe).- If you wish to use the secondary computer for your imageprocessing, please type “Simulator” beside your name in the“Summary” box.C OMMON QUESTIONS:1. Why do I not see anything though eyepieces?- Did you pull out transmitted light detector?2. Why do I not see anything in Laser Mode?- Did you pull out Switch rod?- Did you set Fluorescent filter to 4 (scan)?3. Why is intensity very low in laser mode?- Did you pull out analyzer of DIC?- Did you open lens aperture (40x, 63x, 100x)?Confocal Laser Scanning Microscope Instructions StartupStart-upin order:1. UV lamp: ON [warm up for 10 min] *2. Microscope Main Switch: ON3. Laser fan button: ON (→ check for air that comes from cooling fan behind the door.)4. Scanner power button: ON [wait for 20 sec before starting software]5. PC power button: ON6. Ar/ArKr laser key: ON [warm up for 30 min, best after 1 hr]*7. GreenHeNe laser key: ON [warm up for 15 min]*8. HeNe key: ON [warm up for 15 min]*9. Windows NT login: Ctrl+Alt+Delete- Network: check “Workstation Only”- PC: login name**, TCS_User [password is NOT required]10. Leica Confocal software: start → PersonalAdditional Information*: You can use immediately after the switches are on, but you will not get the best performance of the lasers and UV lamp.**: Each laboratory will be given its own login name and a password.ShutdownShut down in order:1. Ar/ArKr laser key: OFF (start to count for 15 min)2. GreenHeNe key: OFF3. HeNe keys: OFF4. Leica Confocal Software: close5. Scanner power button: OFF< Make a backup CD of your data by “Easy CD creator” >6. Windows: shut down → wait for the message “It is now safe to turn off your computer”7. PC power button: OFF< Lens Cleaning >8. Microscope: OFF9. UV lamp: OFF10. Wait for 15 min (after Ar key OFF) till air from fan is cooled →Laser button: OFFIMPORTANT!- If somebody will use the microscope within 2 hr after you, do not turn OFF the Ar/ArKr laser key(1), Laser button (10), and UV lamp. All the other units should be shut down.- The Mercury lamp of the epifluorescence cannot be restarted within 30 min after use.- Back up your files on CD. Do not leave the files in the workstation.Leica DMR Microscope: General Information & CareLenses:ObjCond Obj Cond Obj Cond 10x ---with & without BFH------------3 – 420x --- 0.17 (1.5) BF H --- --- --- --- 1.2 – 1.3 40x OIL 0.17 (1.5) BFH------H30.4 – 0.563x OIL 0.17 (1.5) BF H D 63 --- --- 0.3 – 0.4 100x OIL0.17 (1.5)BF H D 100 --- --- 0.3 – 0.4Immersion oil:Use only the immersion oil provided with the microscope. Do not mix the immersion oil with other substances including immersion oil from another source. Remove with Windex any oil remaining on the cover slip used in previous observations with a different microscope.Cover slip:Sealing coverslip completely with nail polish or other sealing materials is highly recommended. This prevents mixing of immersion oil and embedding materials, movement of the coverslip when moving the stage, and moving embedded materials during z-sectioning. Be sure that any nail polish is completely dried before mounting slide onto the stage.Lens care:After use, clean lenses only with lens paper. Do not use Kim Wipes, cloth, etc. Fold the paper, and hold both sides of the paper keeping the folded edge strait. Rub the folded edge back and forth over the lens surface. Change the place on the paper and rub until there is no longer evidence of oil on the paper.LensesImmer-sionCoverslip µm (#)Bright FieldDICPhase ContrastStep Size (µm)Galvo stage:Inside frame of the specimen holder is used for z-position setting. Treat it with great care and avoid from bumping it, especially the projecting part of the holder.Automotive stage:A: stepwidth at fine focusing (1=0.1 µm, 2=0.7 µm, 3=1.5 µm)B: motor drive – UP till upper thresholdC: motor drive – DOWND: focusing wheelE: upper thresholdF: lower thresholdTo carry out the following tasks:Set and delete a threshold: pressing and sustaining the E or F keys.Override an upper threshold: with the focusing wheel.Switch between fine and coarse stepwidth: pressing B and C keys, simultaneously.IMPORTANT!- Do not use automotive stage without upper threshold setting: the stage travels to the mechanical end-switch position, which damages the condenser, the objectives and the specimens.- Upper threshold is important for proper application of software (Galvo stage).Koehler illumination setting:1) Focusing image2) Open Aperture diaphragm (13)3) Close Field diaphragm (14)4) Adjust condenser height till the edge of the filter becomes sharp.5) Center Field diaphragm by screws under condenser.6) Open Field diaphragm (14) until it just disappears from the field of view.Microscope setting for:Bright Field (BF), and Epifluorescence (Ef) observations with eyepieces and laser mode• 10x, 20x, 40x, 63x, & 100x lensesBF Ef Laser1. Switch rod PUSH IN PUSH IN PULL OUT2. Halogen lamp switch ON ON ON3. Transmitted laser detector bar PUSH OUT PUSH IN PUSH IN4. Fluorescent light CLOSE (UP) OPEN (DOWN) CLOSE (UP)5. Reflector turret (Fluorescent filter)4 (scan)1, 2, or 3(UV, G, or B)4 (scan)6. Turret for objective side WallastonprismsBF - BF7. Condenser turret H - H8. Polarizer - - -9. Analyzer for DIC PULL OUT PULL OUT PULL OUT10. Fine adjustment of objective sideWallaston prisms- - -11. Illumination intensity control ADJUST MINIMIZE MINIMIZE12. ND filter OFF - -Microscope setting for:Differential Interference Contrast <Nomarski> (DIC) and Epifluorescence (Ef) observations with eyepieces and laser mode• 63x & 100x lenses1) Take out when either 100x objective with 2x zoom or 63x objective with 5x zoom is used.DIC EfLaser1. Switch rodPUSH IN PUSH IN PULL OUT 2. Halogen lamp switchON ON ON3. Transmitted laser detector bar PUSH OUT PUSH IN PUSH IN4. Fluorescent lightCLOSE (UP) OPEN (DOWN) CLOSE (UP) 5. Reflector turret (Fluorescent filter) 4 (scan)1, 2, or 3 (UV, G, or B)4 (scan)6. Turret for objective side Wallaston prismsD - D 1)7. Condenser turret 63 for 63x 100 for 100x- 63 for 63x 100 for 100x8. Polarizer IN - IN 9. Analyzer for DICPUSH INPULL OUTPULL OUT10. Fine adjustment of objective side Wallaston prismsADJUST - ADJUST 11. Illumination intensity control ADJUST MINIMIZEMINIMIZE12. ND filter OFF--Microscope setting for:Phase contrast (Ph), Epifluorescence (Ef)observation with eyepieces and laser mode• 40x lensPh EfLaser1. Switch rod PUSH IN PUSH IN PULL OUT2. Halogen lamp switch ON ON ON3. Transmitted laser detector bar PUSH OUT PUSH IN PUSH IN4. Fluorescent light CLOSE (UP) OPEN (DOWN) CLOSE (UP)5. Reflector turret (Fluorescent filter)4 (scan)1, 2, or 3(UV, G, or B)4 (scan)6. Turret for objective side WallastonprismsBF - BF7. Condenser turret 3 - 38. Polarizer OUT - OUT9. Analyzer for DIC PULL OUT PULL OUT PULL OUT10. Fine adjustment of objective sideWallaston prisms- - -11. Illumination intensity control ADJUST MINIMIZE MINIMIZE12. ND filter OFF - -Leica Confocal Software operation!Focus your sample after the software opens. The software calibrates the Galvo stage during initializing the scanner that causes the focus change.Acquisition Dialog Window1. Press the “Obj.” button and select the objective lens to be used in the open dialog window.Check objective lens every time you switch objectives.2. Press the “Beam” button and select a method in the open “Beam Path Setting”dialog window.A method is a set of hardware settings specifically identifying a certain acquisitiontechnique and a special type of preparation.It is always possible to define and store your own methods in addition to the factory-set methods.3. Press the “Mode” button and select the scan mode.xyz, xzy: spatial scan modext, xyt, xzt, xyzt: time scan modexyλ, xzλ: spectral scan mode4. Press “Format” button to select the scan format.Besides the numerical aperture of the objective and the excitation wavelength, the scanformat, together with the electronic zoom, determines in large part the spatial resolutionof the recorded data. (See Table 1)A structure can only be scanned without information loss if the smallest optically resolvabledistance (=lateral resolution) is scanned with about 2 to 3 raster points.Excitation at 543ObjectiveMagnificationNumerical Aperture Resolution (nm)Scan Format (# x #)Zoom Factors Scan fieldSize (mm)Voxel 1)Size (nm)(current raster distance)Resolution / Voxel Size 2)10 0.3 747 512 1 1500 2930 0.3 747 512 2 750 1465 0.5 747 512 4 375 732 1.0 747 512 6 250 488 1.5 747 512 8 188 366 2.0 10 0.3 747 1024 1 1500 1465 0.5 747 1024 2 750 732 1.0 747 1024 4 375 366 2.0 10 0.3 747 2048 1 1500 732 1.0 747 2048 2 750 366 2.0 10 0.3 747 4096 1 1500 366 2.0 20 0.5 434 512 1 750 1465 0.3 434 512 2 375 732 0.6 434 512 4 188 366 1.2 434 512 8 94 183 2.4 20 0.5 434 1024 1 750 732 0.6 434 1024 2 375 366 1.2 434 1024 4 188 183 2.4 20 0.5 434 2048 1 750 366 1.2 434 2048 2 375 183 2.4 40 1 217 512 1 375 732 0.3 217 512 2 188 366 0.6 217 512 4 94 183 1.2 217 512 6 63 122 1.8 217 512 8 47 92 2.4 40 1 217 1024 1 375 366 0.6 217 1024 2 188 183 1.2 217 1024 4 94 92 2.4 40 1 217 2048 1 375 183 1.2 217 2048 2 188 92 2.4 40 1 217 4096 1 375 92 2.4 63 1.32 – 0.6 165 512 1 238 465 0.4 165 512 2 119 232 0.7 165 512 4 60 116 1.4 165 512 6 40 77 2.1 63 1.32 – 0.6 165 1024 1 238 232 0.7 165 1024 2 119 116 1.4 165 1024 3 79 77 2.1 63 1.32 – 0.6 165 2048 1 238 116 1.4 165 2048 2 119 58 2.8 100 1.4 – 0.7 155 512 1 150 293 0.5 155 512 2 75 146 1.1 155 512 4 38 73 2.1 100 1.4 – 0.7 155 1024 1 150 146 1.1 155 1024 2 75 73 2.1 100 1.4 – 0.7 155 20481150 73 2.11) Volume and pixel ! Voxel2) Resolution / Voxel Size < 2 ! undersamplingResolution / Voxel Size > 3 ! oversamplingExcitation at 488ObjectiveScan Format (# x #)Zoom FactorsScan fieldSize (mm)Voxel 1)Size (nm)(current rasterdistance)Resolution / Voxel Size* Magnification Numerical ResolutionLateral resolution =Numerical aperture0.4 x λRaster point distance(= Scan Frequency) (= Voxel size)Format size Scan field size =10 0.3 651 512 1 1500 2930 0.2651 512 2 750 1465 0.4651 512 4 375 732 0.9651 512 6 250 488 1.3651 512 8 188 366 1.8651 512 10 150 293 2.2 10 0.3 651 1024 1 1500 1465 0.4651 1024 2 750 732 0.9651 1024 4 375 366 1.8651 1024 6 250 244 2.7 10 0.3 651 2048 1 1500 732 0.9651 2048 2 750 366 1.8651 2048 3 500 244 2.7 10 0.3 651 4096 1 1500 366 1.8651 4096 2 750 183 3.6 20 0.5 390 512 1 750 1465 0.3390 512 2 375 732 0.5390 512 4 188 366 1.1390 512 8 94 183 2.1 20 0.5 390 1024 1 750 732 0.5390 1024 2 375 366 1.1390 1024 4 188 183 2.1 20 0.5 390 2048 1 750 366 1.1390 2048 2 375 183 2.1 40 1 195 512 1 375 732 0.3195 512 2 188 366 0.5195 512 4 94 183 1.1195 512 6 63 122 1.6195 512 8 47 92 2.1 40 1 195 1024 1 375 366 0.5195 1024 2 188 183 1.1195 1024 4 94 92 2.1 40 1 195 2048 1 375 183 1.1195 2048 2 188 92 2.14096 1 375 92 2.1 40 1 19563 1.32 – 0.6 148 512 1 238 465 0.3148 512 2 119 232 0.6148 512 4 60 116 1.3148 512 6 40 77 1.9148 512 8 30 58 2.5 63 1.32 – 0.6 148 1024 1 238 232 0.6148 1024 2 119 116 1.3148 1024 3 79 77 1.9148 1024 4 60 58 2.5 63 1.32 – 0.6 148 2048 1 238 116 1.3148 2048 2 119 58 2.5 63 1.32 – 0.6 148 4096 1 238 58 2.5 100 1.4 – 0.7 139 512 1 150 293 0.5139 512 2 75 146 1.0139 512 4 38 73 1.9139 512 6 25 49 2.9 100 1.4 – 0.7 139 1024 1 150 146 1.0139 1024 2 75 73 1.9139 1024 3 50 49 2.9 100 1.4 – 0.7 139 2048 1 150 73 1.9139 2048 2 75 37 3.85. Press “Speed” button to select the scan speed.The higher the set scan speed, the shorter the dwell time of the laser point.The higher the scan format at a constant speed, the shorter the dwell time of the laser pointover a sampling point.Using a lower scan speed results in a better signal-noise ratio, but relatively long impact of the light on the specimen can bleach it photochemically.6. Press “Pinh” button to set the detection Pinhole. Click the Airy 1 button, the detection pinhole is set automatically to the optimum value of 1 Airy unit depending on the objective in use. In addition to the numerical aperture of the objective and the wavelength of the light, the detection pinhole also determines the thickness of the optical sections.The wider the diameter of the pinhole, the more light reaches the detector. The imagebecomes brighter but blurring from structures outside the focal plane will also appear in theimage, making it increasingly unfocussed. Increasing the diameter of the pinhole above 1 Airy unit is recommended only for detecting very weak signals.7. Press “Continuous” button to start the continuous scan.Use the control panel and its defined acquisition parameters which include:a. Z-positionb. Amplification factor of the selected detectorc. Zoom (See Table 1)8. Click “Signal” button for setting the Detectors: Gain value and Offset value.1) Stop scanning.2) Click the color look-up tables (Arrows) currently working.3) Select Glow Under and Over.4) Adjust Gain value using PMT Gain Dials on the panel box or sliders in the SignalWindow as blue color scatters a little. Blue color indicates the strongest signal.5) Adjust Offset value using PMT Offset Dials or sliders in the Signal Window asgreen color uniformly covers an image (Value shouldbe 0 or -1). If you are working with two detectors theratio of green color in the two views is adjusted to bethe same.Detectors set for eliminating “Cross-talk” arenow set.9. Stop the continuous scan by pressing the “Cont.”button.10. Press “Aver.” button to define the number ofsampling times (frame average).Large number of averaging reduces noisebut can cause fading.11. Press “Single Scan” button to get a single optical image.3D Image3. Turn “Z-Pos” Dial on Panel Box.4. Press the “Begin” button to define the starting position of the 3D series.5. Press the “End” button to define the end position of the 3D series.6. Stop the continuous scan by pressing the “Cont.” button.7. Press “Sect.” button to define the number of optical sections. Go to “Others” to set by option.Image Dim. z/y (mm): The height of the entire image stack betweenthe beginning and end points of the image series.# Sections: The number of configured sections.Step size (mm): The step size, i.e. distance between two sections.Calculate the number of sections / Unchanged the height of the image stack Enter the desired step size in the “Step Size” field.Then click the “Calculate” button next to the Step Size filed.Calculate the number of sections / Unchanged the number of sections:Enter the desired step size in the “Step Size” field.Then click the “Calculate” button next to the # of Sections field.Calculate the step width / Unchanged the height of the image stack:Enter the # of desired sections in the # Sections” field.Then click the “Calculate” button next to the # Sections filed.Switching z-scan Mode:1. Press “z-Scan” button and switch themode.2. Press “z Position” on Panel Box Setting DialogWindow to switch over the modes.Galvo Z-WideMinimum step width 0.04 µm0.1 µmMaximum drive distance 170 µm Maximum drive distance of stage (cm)Drive module Galvo stage Microscope side motor drive: StageCalculate the step width / Unchanged the number of sections:Enter the number of desired sections in the # Sections” field.Then click the “Calculate” button next to the Step Size filed8. Press “Series” button to create an image series.When working with specimens that are:Low signal Bleach-sensitive Detector Gain ↑Rise Scan time ↓ReduceLaser power ↑Rise↓ ReducePinhole ↑ OpenAveraging ↓ Reduce or no useScan Speed ↓ Slow (200)↑ Fast (800)Scan Format ↑ Avoid undersampling↑Avoid oversamplingZoom ↓Short intervalBeam expander ↑ Use small diameter (3)↓ Use large diameter (6) Accumulation + Useful(+ Useful)Data HandlingFile types used in this applicationExperiment (*.lei) A Leica-specific, binary data format. When the experiment isloaded, both the image data and the experiment settings areloaded.Annotation sheet (*.ano) A Leica-specific, binary data format. The elements on theannotation sheet, such as images, texts and graphic images,are each available as individual objects.Tiff files (*.tif) These are Leica image files in single and multi Tiff format.Experimental files in RGB-Tiff format can be loaded as well.I. Experiment Overview Dialog WindowIMPORTANT:Experiment Dialog Window shows files thatare temporarily stored on RAM memory.Experiment: Click the “New” button forcreating a new Viewer window, which createsa new experiment. An experiment is a file that consists of one or more individual images or image series. This allows you to keep several images, each record using different scan parameters, or edited versions of images in one experiment.File name: Unsaved directories are named as “Experiment #”.Active mark (red check): This mark shows a viewer currently activated. Activate viewer by clicking a viewer or the image file on the directory.Series #: A new Series file is added in a new experiment or a directory that contains an activated image each time you press the “Series” button.Image #: A new image file is created in a new experiment or a directory that contains an activate image each time you press the “Single Scan” button.Preview #: When you press “Cont.” button, an image file is created in a new experiment or a directory that contains an activated image or series file. When a “Preview file” is activated and you do a continuous scanning, the preview file will be overridden.CLSM INSTRUCTION Ver. 2; CBS; U of G II. Saving Images1. Activate a view you want to save.2. Click the “Save as” button to save the data of the current experiment (*.lei) or the current annotation(*.ano) sheet.3. When saving an experiment, a folder is created at the file level with the name of the experiment. Thisfolder then contains the description file (*.lei) for the experiment as well as the individual image files.The description file (*.lei) contains parameter settings and color information for each image belonging to the experiment.When saving an experiment, the new folder contains all images belonging to the directory. Make sure to delete unnecessary images.IMPORTANT: the original contents (*.lei, *.tif) in a folder need to be a set. Keep the file size within the memory size of a CD (i.e. 640 MB) for your backup.RECOMMENDED: save one original image (Series or Image) in one folder (one Experiment) in which the processed images (i.e. 3D images) can be contained.AMPLIFICATION OF EACH IMAGE: right click of the image in the Experiment Overview WindowIII. Saving an image from a viewer as a hard copy1. Right click on the viewer and select “Send to” then “Experiment”2. Select one from three types as follows:a. Selection (raw): send raw data of only selected image on a viewer. Additives onthe image such as scale bar, numbers and letters will not be sent.b. Selection (snapshot): send all information with additives on the selected image on aviewer.c. All (snapshot): send all images with additives in a viewer.3. Click “Save” button to save the change of the experiment.IMPORTANT: Display Zoom affects for “Snapshot”. Select “Auto” or “1 : 1” before images are sent.IV. Saving an image as an animation file1. Create a 3D image from an image series.All processed images are stored on RAM. Delete unnecessary images!2. Right click at a 3D image and select “Export”.3. Put file name and save as a file type [*.avi].4. Choose “uncompressed”.Page 21 of 21About WebCT | Help | Students | Faculty & Designers | Guests | Site MapNewsWelcome to Courselink -- the University's portal to on-campus courses using WebCT.To see a list of classes that use WebCT Click Here.Some users have reported a "Permission Error" or a Search Page () appearing when they click on "Login to WebCT" if this is the case see our FAQ by clicking HereIf you need to register for a class at the University of Guelph you should use Webadvisor .If you need information on Distance Education then you should consult Open Learning .See FAQ's for more helpContact WebCT Support(click to login to WebCT)WebCT Login Instructions q Courses That Use WebCT q Look for my Distance Educationcourseq Test My Browser Setup q Email Courseware Support q WebDAV Instructions (drag anddrop files and folders into your course files)q Enhancing On-campus Courses Through Web-based LearningAbout This Site | Copyright InformationCourselink At Guelphhttp://courselink.uoguelph.ca/ [2/26/2004 11:14:55 AM]。
激光共聚焦显微镜操作手册最终版
徐珏2011.12.3 17:59 于苏州宝石御景园目录标准探测模式（DU4）图像拍摄流程 (1)一、软件开启 (1)二、基本设置 (2)[A1 Setting]界面主要功能区域简介 (2)[Setting]界面功能简介 (4)[Filter and Dye]功能区简介 (6)串色现象解释及Line模式图解 (6)Line模式图解 (7)[scan setting]功能区简介 (7)三、获得图像 (8)[Acquisition]功能区简介 (10)放大方式选择按钮简介 (13)图片扫描参数调用简介 (17)JP2格式图片转化为JPG/TIFF格式图片的操作 (18)各通道荧光图像自由叠加的介绍 (18)数据分析 (21)一、ROI区域分析 (21)二、LUTs调节图像荧光亮度 (23)三、去背景 (25)四、标尺添加 (26)六、长度面积等常规测量 (28)七、细胞计数 (28)[Binary Toolbar]界面简介 (32)光谱扫描模式（SD模式） (34)一、SD模式基本设置 (34)[Detector]标签页功能简介 (35)[Binng/Skip]标签页简介 (36)二、图像获得 (37)三、数据分析 (39)[Spectrum Profile]视窗简介 (40)四、光谱拆分 (40)虚拟滤光扫描模式（VF模式） (47)一、VF模式基本设置 (47)[Detector]设置标签页简介 (48)[Gating Setting]标签页简介 (48)二、获得图像 (48)时间序列拍摄 (49)[Capture Timelapse]视窗简介 (49)[ND]界面简介 (51)[Time Measurement]界面简介 (53)[Capture Z-series]视窗简介 (55)拼大图拍摄 (60)光活化序列拍摄 (61)[Photo Acquition]功能区简介 (62)[Sequential Stimulation]界面简介 (63)常见细胞类型和细胞器形状 (65)常用荧光染料及应用领域 (68)标准探测模式（DU4）图像拍摄流程一、软件开启1、双击桌面NIS-Element图标。
激光共聚焦显微镜操作流程
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文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor. I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!激光共聚焦显微镜操作流程如下：1. 开启显微镜电源和激光电源，等待仪器预热。
激光共聚焦操作流程
激光共聚焦操作流程英文回答：Confocal Laser Scanning Microscopy (CLSM)。
Introduction:Confocal laser scanning microscopy (CLSM) is a powerful imaging technique that allows for the acquisition of high-resolution, three-dimensional images of biological samples. CLSM utilizes a laser beam to scan the sample, and the emitted fluorescence is detected by a photomultiplier tube (PMT). The resulting images provide detailed information about the structure and organization of cells and tissues.Procedure:1. Sample Preparation:Biological samples are typically fixed and stainedwith fluorescent dyes to enhance their visibility.The samples are then mounted on a glass slide and coverslipped.2. Microscope Setup:The CLSM system consists of a laser, scanning mirrors, a PMT, and a computer.The laser is focused onto the sample using an objective lens, and the scanning mirrors direct the beam to different points of the sample.3. Image Acquisition:The laser scans the sample in a raster pattern, and the emitted fluorescence is detected by the PMT.The intensity of the fluorescence is recorded at each point, and this data is used to generate an image.4. Image Processing:The raw image data is processed to remove noise and enhance the contrast.Three-dimensional images can be generated by stacking multiple two-dimensional images acquired at different depths within the sample.5. Analysis:The processed images can be analyzed to quantify various parameters, such as fluorescence intensity, cell size, and tissue architecture.Advantages of CLSM:High resolution and detail: CLSM provides high-resolution images with a lateral resolution of approximately 200 nm and an axial resolution of around 500 nm.Three-dimensional imaging: CLSM allows for the acquisition of three-dimensional images, which provides a more comprehensive view of the sample.Non-invasive: CLSM is a non-invasive technique that does not damage the sample.Versatile applications: CLSM can be used to image a wide range of biological samples, including cells, tissues, and embryos.Limitations of CLSM:Photobleaching: Fluorescent dyes can be photobleached by excessive laser exposure, which can limit the duration of imaging sessions.Autofluorescence: Some biological samples exhibit autofluorescence, which can interfere with the detection of specific fluorescent dyes.Limited penetration depth: The penetration depth ofthe laser beam is limited, which can restrict imaging to the superficial layers of thick samples.中文回答：激光共聚焦扫描显微镜（CLSM）。
激光共聚焦显微镜操作方法-confocol
(1)用室温下的PBS洗涤细胞三次，弃去 PBS;
(2)用4%的多聚甲醛溶液 37o C固定细胞15-30min ;
⑶用PBS洗三次，每次 5min；
(4)用 0.2%的 TritonX-100 透化细胞 10min ;
(5)PBS洗三次，每次 5min ;
(6)用含1%的二抗来源血清(消除非特异性影响 ^^PBS封闭1h;
(7)PBS洗三次，每次 5min ;
(8)室温孵育一抗1h(用封闭液配制，1:50);(两个抗体可以一起孵育)
(9)PBS洗三次，每次 5min ;
(10)室温避光孵育二抗 1h(用封闭液配制，1:50);
(11)用PBS洗三次，每次5min (以下步骤均需避光进行)；
(可省略)(12)用RNase在37o C下消化细胞 30min(使用终浓度为 20 口 g/ml以除去细胞内的
RNA ;
.- --. . -------. . ■.'V-* ■ ■ ,
(13)PBS洗三次，每次 5min ;
(14)用Hochest33342对细胞核进行染色(使用浓度为1科g/m1:1000),室温避光处理20min ;
(15)用PBS洗三次，每次 5min；
(16)置于激光共聚焦才3描显微镜下，用100汹由镜进彳T观察，并保存结果。
激光共聚焦显微镜使用流程
激光共聚焦显微镜使用流程
1.导师网络注册并等待审核（导师注册过的跳过此步骤）
2.学生注册，注册时选定自己的导师
3.带本人校园卡及导师签名的注册申请表到先知楼603室
审核并激活校园卡
4.转账、系统充值
5.参加激光共聚焦显微镜使用培训，取得上机操作资格。
6.网络预约时间使用，并等待审核。
7.按时上机使用，使用刷卡开机。
8.结束使用，按操作规程关机，并刷卡。
注：
1.每一期共聚焦显微镜培训分两次，每次约
2.5小时，每两周进行一期。
具体时间看网站通知。
2.共聚焦显微镜开放时间9:30-19:00
3.本人预约，仅限本人使用，不得用他人校园卡上机。
4.预约成功后，可以用校园卡刷卡进先知楼603西门。
5.刷卡开机使用，使用完成关机后务必再次刷卡结束使用，以免造成多
计费。
分析测试中心
2013-04-22。
共聚焦操作流程
一、实验环境观察仪器是否有外表明显损坏，如需开启空调，一般控制在25度。
拉上窗帘，避免标本荧光淬灭及杂光干扰。
二、开机查看实验记录，确认是否马上能开机（注意：汞灯必须在关机20分钟以后才能再开启，一般为30分钟）。
确认后可执行以下操作：（务必先开电源，后开钥匙！！！）。
1、打开PSU（主电源）2、打开激光器（只开启需使用的激光器）1）、氩离子多线激光器（458/488/515，应用染料：GFP/FITC/Fluo3/Alexa488/CFP/YFP等）。
2）MCPSU半导体综合激光器（LD405应用染料：DAPI/Hoechst/BFP等；LD635应用染料：CY5/ Alexa647等）。
3）LD559激光器（注意：电源打开后不要马上开钥匙，等待TEMP指示灯不闪烁后，再开钥匙，红色指示灯不闪烁后，可正常使用）。
3、打开显微镜电源IX2-UCB4、打开荧光汞灯（注意：开启后，“15分钟内勿关”）5、打开电脑主机、显示器，登陆windows xp系统，双击共聚焦软件，打开FV10-ASW应用软件。
三、取图1、DIC（微分干涉差）明场立体图（1）、插入起偏镜、检偏镜（DIC滑块）。
（2）、FV10-ASW软件中点击透射光观察按钮，打开卤素灯快门，使用TD滑块（软件TD lamp项）控制卤素灯的光强，倒置显微镜下观察标本，进行标本聚焦。
1）、使用手控面板选择合适倍率的物镜下观察（注意若用60×油镜时，检偏镜部位要推上弯杆），并检查各物镜对应的DIC元件。
2）、可在检偏镜部位用旋钮进行对比度的调节。
（需要时可使用，一般勿调）（3）、FV10-ASW软件中点击透射光观察按钮，关闭卤素灯快门。
打开DIC通道，执行取图操作。
1）、调节扫描速度至最快（2.0us/pixel），size为512by，点击，进行快速预扫，寻找焦平面。
步骤：适当调节HV、gain及offset参数（一般为能看到图像即可），快速调节寻找焦平面，如需放大光学倍数，可适当调节。
nikon共聚焦显微镜安全操作规程
Nikon 共聚焦显微镜安全操作规程一、操作步骤1、开机：大体按照：电脑→激光台（打开要用的激光按钮）→卤素灯→载物台→显微镜→控制器→软件，荧光灯需要就打开，不需要不用打开2、拍摄流程：打开软件Nikon confocal，ok进入软件操作界面，点eye port，看目镜聚焦，然后点eye port及Interlock解锁切换到激光光路L100，根据自己实验来设置参数和探测方法（DU4），然后点scan，重调Z轴找焦面，对通道进行HV和功率优化，停止scan，点击capture，完成一次拍摄。
3、图像保存：⑴数据保存：File，save as，保存原始数据文件格式.ND2；⑵图片保存：File，Inport/Export，Export ND document，在Export ND document 窗口中Channels，TIF compatibility Options（选择第二个或第三个），保存叠加图像时，在Export ND document 窗口中Channels勾选Insert Overlaps，再点击export4、关机：Z轴降到500以下，物镜转到空挡，最后与开机完全相反顺序进行关机。
二、注意事项：1、放置样品时，确保样品的中心区域在物镜的正上方，且尽量要物镜在两个夹片中心区域，以免转换物镜时，撞坏物镜；2、原则上所有的光源都非常忌讳频繁开关，会大大缩短其使用寿命。
荧光灯、卤素灯：开机后不管是否使用，都最好半小时后再关闭；关机后至少等十五分钟再开机。
激光：开机后不管是否使用，都最好一小时后后关闭；关机后至少等一小时再开机。
3、明场卤素灯的功率值不能超过4，否则会导致温度过高烧坏光纤，影响明场成像；4、60倍和100倍物镜是油镜，滴油时注意只需沾一下，切勿多，滴了油，如果要转物镜，必须先将当前滴了油的物镜用石油醚清洗擦干净才能转换；5、实验结束或者换样品转换镜头时必须按照以下程序进行操作：首先，取下样品，将物镜Z轴值降到500以下，然后，将载物台的载玻片拨至两端，最后再进行物镜的转换。
共聚焦显微拍摄的步骤及方法
共聚焦显微拍摄的步骤及方法
一、技术简介
激光扫描共聚焦显微镜是在荧光显微镜成像基础上加装了激光扫描装置，利用计算机进行图像处理，把光学成像的分辨率提高了30%-40%，使用紫外或可见光激发荧光探针，从而得到细胞或组织内部微细结构的荧光图像，在亚细胞水平上观察诸如Ca2+ 、PH值，膜电位等生理信号及细胞形态的变化，成为形态学，分子生物学，神经科学，药理学，遗传学等领域中新一代强有力的研究工具。
激光扫描共聚焦显微镜以激光作为光源，激光器发出的激光通过照明针孔形成点光源，经过透镜、分光镜形成平行光后，再通过物镜聚焦在样品上，并对样品内聚焦平面上的每一点进行扫描。
样品被激光激发后的出射光波长比入射光长，可通过分光镜，经过透镜再次聚焦，到达探测针孔处，被后续的光电倍增管检测到，并在显示器上成像，得到所需的荧光图像，而非聚焦光线被探测针孔光栏阻挡，不能通过探测针孔，因而不能在显示器上显出荧光信号。
二、实验流程
1. 样品的固定（多聚甲醛固定法）。
2. 组织的冷冻包埋和切片（干冰冷冻包埋法）。
3. 免疫荧光标记。
4. 先在荧光显微镜下观察确认标记成功，然后移至共聚焦显微镜下进行观测。
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(1)用室温下的PBS洗涤细胞三次，弃去PBS；
(2)用4%的多聚甲醛溶液37o C固定细胞15-30min；
(3)用PBS洗三次，每次5min；
(4)用0.2%的TritonX-100透化细胞10min；
(5)PBS洗三次，每次5min；
(6)用含1%的二抗来源血清(消除非特异性影响)的PBS封闭1h；
(7)PBS洗三次，每次5min；
(8)室温孵育一抗1h(用封闭液配制,1:50)；(两个抗体可以一起孵育)
(9)PBS洗三次，每次5min；
(10)室温避光孵育二抗1h(用封闭液配制,1:50)；
(11)用PBS洗三次，每次5min (以下步骤均需避光进行) ；
(可省略)(12)用RNase在37o C下消化细胞30min(使用终浓度为20μg/ml)以除去细胞内的RNA；
(13)PBS洗三次，每次5min；
(14)用Hochest33342对细胞核进行染色(使用浓度为1μg/ml,1:1000),室温避光处理20min；
(15)用PBS洗三次，每次5min；
(16)置于激光共聚焦扫描显微镜下,用100×油镜进行观察,并保存结果。