ImageJ软件在显微图像细胞计数中应用的可行性研究
何宝林;杨晚竹;陈婷;郑昭烽;林芳珍;李迎辉;高瀛岱
【期刊名称】《医疗卫生装备》
【年(卷),期】2022(43)2
【摘 要】目的:探究开源软件ImageJ在显微图像细胞计数中应用的可行性。方法:使用ImageJ软件对荧光染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)标记的悬浮细胞和羧基荧光素二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein diacetate succinimidyl ester,CFSE)标记的半贴壁细胞进行手动计数和自动计数,对脂肪包备蛋白Perilipin标记的脂肪细胞进行手动计数并通过编写程序对其进行自动计数,分别统计2种计数方法的准确率(以手动计数作为金标准)和计数时长。结果:在计数准确率方面,悬浮细胞自动计数的准确率为(98.28±1.59)%,半贴壁细胞自动计数的准确率为(97.43±0.44)%,脂肪细胞自动计数的准确率为(94.11±0.76)%。在计数时长方面,悬浮细胞自动计数较手动计数耗时长[(225.56±7.83)s vs(185.45±8.09)s],P<0.05;半贴壁细胞和脂肪细胞自动计数时长明显短于手动计数时长[(65.66±2.01)s vs(550.10±18.01)s,(218.33±6.38)s
vs(776.04±8.72)s],P均<0.05。结论:ImageJ软件能够实现多种类型显微图像的细胞计数,还可兼容各种标记类型细胞显微图像的自主分析,为科研人员的图像分析工作提供了便利。
【总页数】7页(P13-18)
【作 者】何宝林;杨晚竹;陈婷;郑昭烽;林芳珍;李迎辉;高瀛岱
【作者单位】中国医学科学院血液病医院 【正文语种】中 文
【中图分类】R318;TP317.4
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利用ImageXpress Micro进行长时间活细胞成像和心肌细胞功能评价
活细胞成像技术是最近几年兴起的一项技术，能够在细胞接近生理的状态下观察细胞形
态改变和蛋白的表达，该技术能够避免传统采用固定细胞或组织的研究方法中，固定细胞过
程中造成的细胞形态的改变和结构改变，能够更加真实的反映出细胞的特性，因而广受推崇。
MD ImageXpress Micor高内涵系统具有多种特性能够进行长达数天的活细胞的观察，同时，
由于采用著名的MD软件产品MetaMorph为基础的MetaXpress高内涵分析软件，具有极高
的灵活性和扩展能力，除活细胞成像之外还能够能够对某些细胞（如心肌细胞）的生理药理
特性进行评价。
神经细胞长时间成像
神经细胞是公认的较为敏感和脆弱的细胞，对于外界环境较为敏感。对于活的神经细胞
的长时间培养和观察具有较大困难。采用ImageXpress Micro系统能够实现神经细胞的长时
间观察培养。 方法：
从新生大鼠大脑海马获得约1立方毫米大小组织块。移入含有0.25%胰酶的离心管中
37℃，5%CO2 孵育箱内消化20～30 min。加入含胎牛血清的培养液终止消化，离心重悬细
胞，按0.6×105/ml 铺于96 孔板中。24 h 后换为NeurolbasalMedium/B27 无血清培养液，
以后每2~3 d 半量换液1 次。7 d 后获得原代神经细胞。
图像获取：
将样品放入有环境控制功能的MD ImageXpress Micro高内涵系统内，采用激光自动聚
焦进行样品聚焦，在10X物镜下每隔5分钟进行一次相差图像拍摄，结果如下：
图1. 用ImageXpress Micro进行神经细胞的长时间观察培养，观察时长50小时
小结：
ImageXpress Micro采用的环境控制系统能够维持细胞所需的温度、湿度和CO2条件，
其中温度为细胞的酶活性提供了温度保障，确保细胞的生理机能，湿度维持能够保证细胞外
酶标仪成像功能检测手册
目录目录
一、具有成像功能的多功能酶标仪基础................................................................1 1.1、什么是多功能酶标仪..............................................................................1 1.2 、具有成像功能的多功能酶标仪...............................................................1 1.3 、酶标仪成像功能的优势..........................................................................1二、成像功能硬件特点..........................................................................................3三、酶标仪成像功能的应用分类...........................................................................4四、酶标仪成像功能的应用举例...........................................................................5 4.1、透射光通道下的无标记细胞识别和计数.................................................5 4.1.1、StainFree无标记细胞计数............................................................5 4.1.2、DAPI染色替代方案：活细胞成像计数...........................................6 4.1.3、无标记细胞增殖和形态评价..........................................................9 4.2、荧光蛋白表达.......................................................................................11 4.2.1、GFP的转染优化..........................................................................11 4.3、细胞活性/凋亡/毒性检测......................................................................15 4.3.1、心肌细胞毒性评价......................................................................16 4.3.2、细胞热激应答.............................................................................19 4.3.3、诱导多能干细胞来源的细胞毒性测定.........................................22 4.3.4、用SpectraMaxMiniMax细胞成像系统和 MetaMorph软件测量神经突触...................................................25 4.3.5、GM-CSF和TNFα诱导的细胞凋亡检测.........................................29 4.4、玻片及大样本成像................................................................................32 4.4.1、荧光细胞爬片和组织切片...........................................................32五、MiniMax成像参数设置和图像采集流程........................................................35 5.1、选择成像功能.......................................................................................35 5.2、选择孔板类型及读板区域.....................................................................35 5.3、选择成像视野数...................................................................................36 5.4、设置采集曝光时间和聚焦.....................................................................36 5.5、图像分析设置.......................................................................................37 5.6、数据测量..............................................................................................37 5.7、感兴趣区域设置...................................................................................38
Quick Start Guide ImageXpress® Micro Confocal & MetaXpress® 6 Page 1 of 13 The purpose of this guide is to briefly describe: I. Turn on system and acquire plate with saved settings II. Test acquisition settings III. Define new acquisition settings IV. View images and run an analysis I. Turn on System and Acquire Plate with Saved Settings 1. Turn on the system: Light source (if not already on) IXM Power Supply/Options Controller Box (Also controls Transmitted Light, Environmental Control or Fluidics modules) Computer and Monitor 2. Go to the MetaXpress folder and double-click on the appropriate hardware profile shortcut 3. Login to MDCStore database with username and password *NOTE* Your database, username, and password maybe different. Refer to your administrator for this information Username moldev Password moldev 4. If you log in as system administrator (sa), the next window is a warning regarding security risks; click OK 5. Select Group (security level) and click OK Page 2 of 13 6. In the main toolbar, click or in the main menu select Screening > Plate Acquisition Setup 7. To load a previous saved protocol, click on in Plate Acquisition Setup 8. Click to search windows for the appropriate .hts file. If the settings file is saved to the database, highlight the protocol and click 9. Click to open the door and place the plate in the in the system Click to close the door 10. Alternatively, you can use the Main Taskbar to open and close the door. Click Click or 11. On the Run tab, update the folder name, plate name, and description as desired 12. Click to begin acquiring the plate Page 3 of 13 II. Test Acquisition Settings 1. Open Plate Acquisition Setup 2. In the plate and site section of Plate Acquisition Setup, right-click on the desired well and/or site to move the plate to that position (indicated by a dark green color) 3. Test the acquisition settings by clicking to perform a large range autofocus and snap image routine to perform a focus and snap image routine (if Z series has been activated, all planes will be acquired) to perform an autofocus and snap image routine all for all wavelengths (if Z series has been activated, all planes will be acquired) 4. Adjust the acquisition settings, if necessary: Adjust the focus offset by clicking or adjusting the number manually Adjust the exposure time by clicking or changing the number manually *NOTE* Click on the wavelength name to open the corresponding wavelength tab for advanced options 5. When you have optimized settings, click Molecular Devices recommends enabling Click to search for a location on the hard drive. 6. Click to begin acquiring the plate Page 4 of 13 III. Define New Acquisition Settings 1. Open Plate Acquisition Setup 2. Select the Configure tab 3. Select the Objective and Camera tab i. Select the appropriate magnification from the drop-down menu ii. Set binning (2 for cell counting and cell scoring; 1 for fine sub-cellular detail) iii. Select Acquisition Mode: Widefield or Confocal (3 possibilities depending on system configuration: 60 um pinhole, 42 um pinhole, or 50 um slit) 4. Adjust the objective correction collar if necessary (setting on objective should match physical plate bottom thickness in mm X refractive index of material – 1.59 for Plastic, 1.52 for Glass). On the Run a Plate Taskbar, click on to step through the process. 5. Select the Plate tab and select the appropriate plate type from the drop-down list Page 5 of 13 6. Select the Sites to Visit tab and select the appropriate number of sites Single Site: image one site per well in the center Fixed number of sites: image the number of selected sites for every well. Adjust number and spacing of sites. Left-click on sites to select (green) and deselect (grey). Right-click on any site to move the plate to that site position (dark green) Adaptive acquisition: collect the minimum number of sites to image at least the cell count indicated by the user. The Adaptive Acquisition section will appear allowing the user to choose wavelength, size and threshold settings, and desired minimum count for cells Multi-well: collect multiple wells within one image which is then cropped to define single wells automatically Custom field of view (%): reduce the size of each image by the percentage entered. This is useful when the field of view covers more than the site/well area desired 7. Select the Acquisition tab to select Autofocus and Acquisition options 8. Autofocus options: Always select Enable laser-based focusing Enable image-based focusing for thick samples or those with different focal planes from site-to-site or well-to-well 9. Acquisition options: Enable Acquire Time series for timelapse experiments Enable Acquire Z series for Z step acquisition 10. Other options: If running a journal during acquisition, enable this option to activate the Journals tab If an analysis has already been setup, enable Analyze Images After Acquisition *NOTE* this requires an offline computer to be in Auto-run mode or running PowerCore software To correct for uneven background, enable Perform shading correction and select the appropriate directory where shading correction images are saved
