1、高分辨活细胞成像技术交流主要内容1.高分辨活细胞实验的要求高分辨活细胞实验的要求2.高分辨活细胞成像技术的种类以及介绍高分辨活细胞成像技术的种类以及介绍3.DeltaVision Elite高分辨成像系统在植物领域的应用高分辨成像系统在植物领域的应用现有的显微镜技术科学影像技术的三个基本参数科学影像技术的三个基本参数 空间分辨率空间分辨率 时间分辨率时间分辨率 定量分析所需数据通量定量分析所需数据通量.高分辨活细胞成像的要求1.空间分辨率要高（2D&3D)2.时间分辨率根据实验来选择3.保证细胞的活性（降低光毒性，减少荧光淬灭）4.长时间运行仪器各方面的稳定性6/GE Healthcare8/
2、7/2022yx100 nm(0.1 mm)Fluorescent Bead in a Light Microscope显微镜成像的信号分布7/GE Healthcare8/7/2022Point Spread Function(PSF)zx100 nm(0.1 mm)Fluorescent Bead in a Light Microscope8/GE Healthcare8/7/2022如果能看得更清楚共聚焦共聚焦 VS 影影像还原像还原microscopedeconvolutionmicroscopeconfocol10/GE Healthcare8/7/2022三种常见的高分辨活细胞成像
3、技术Light microscopy techniques for live cell imaging Science,2003单击此处编辑母版副标题样式cell objective宽场照明宽场照明Cell Slideobjective激光共聚焦激光共聚焦Slide1.Low intensity(InsightSSI 22-89mW).2.Large illuminating area.3.Weak damage to the cell.Average:52.8mW/cm21.High intensity(Laser 25-250mW).2.Focus into a small volume.
4、3.Significant damage.Average:30000000mW/cm2照明强度比较照明强度比较单击此处编辑母版副标题样式sCMOS极大的提高了成像速度For decades CCDs were a superior choice for fluorescent microscopy because of serious limitations of CMOS detectors:small chip size(0.5”format or less)lower sensitivity(20e-)until scientific grade CMOS(sCMOS)chip was
5、developedNew generation of CMOS cameras offers:Large field-of-view (whole well imaging for 96 well plate at 2X)Higher Sensitivity than scientific CCDs5 x less noise in comparison with industry standard cooled CCDsHigher dynamic range than scientific CCDs Faster frame rates than scientific CCDs Large
6、 Field of ViewLower image noiseCCD sCMOSsCMOS camera essentials:Scientific grade CMOS chip 2560 x 2160 pixels(5.5 Megapixel)1 electron read-noise 33K electron well-depth 50-55%max QE 400fps 512*512sCMOS data and images are taken from PCO.edge sCMOS 单击此处编辑母版副标题样式准确的采集信号，准确的点扩散函数和准确的采集信号，准确的点扩散函数和软件结合
7、才能实软件结合才能实现影像还原现影像还原A:激发光强度激发光强度B：采集：采集PSF，由，由载物台精度决定载物台精度决定C:PSF，由物镜决，由物镜决定定 f(A,B,C)=单击此处编辑母版副标题样式Astigmatism&Coma Spherical Aberration rejectrejectAxial Skew reject Pass all three colors share a common vertex.Pass all three colors overlap to form a single white pixel(hence all four vertices are s
8、hared by all three colors)Fail-One or two of the colors do not share a common vertex.完美的物镜完美的物镜单击此处编辑母版副标题样式镜镜油的选择油的选择长时间实验的系统稳定性因素1.多点实验时XY的漂移电动平台的长时间精度2.焦距的稳定性Z轴稳定系统3.温度敏感细胞的长时间观察温度控制的稳定4.软件的健壮程度Title or Job Number|XX Month 201XGE Business Unit Name16单击此处编辑母版副标题样式Ultimate Focus焦距稳定系焦距稳定系统统正在申请专利正在
9、申请专利无论机械移动或环境温度波动，都能维持无论机械移动或环境温度波动，都能维持Z轴聚焦位置（轴聚焦位置（25nm）持续监控校正持续监控校正Z轴位置，保证各个时间点的数据准确收集轴位置，保证各个时间点的数据准确收集聚焦校正时间聚焦校正时间200ms只和标记点相关，和其他条件物镜、介质、液流等无关只和标记点相关，和其他条件物镜、介质、液流等无关单击此处编辑母版副标题样式Keep your target in focusVero cell transfected with CFP-actinTime laps:12 hrsOne frame/5 minsSingle sliceExposure:2
10、00ms中科院病毒所中科院病毒所XY电动平台的稳定性Pixel=64 nm in x,yOptical Resolution=200 nm in x,y DeltaVision stages confine drift to levels undetectable by the camera for superior time lapse-imagingxy单击此处编辑母版副标题样式 多多点观察以及细胞追踪点观察以及细胞追踪 keep your target in the field of viewx,y=100.0,100.0 x,y=100.0,100.0 x,y=100.0,100.0
11、x,y=40.0,100.0高分辨活细胞成像的要求1.空间分辨率要高（2D&3D)2.时间分辨率根据实验来选择3.保证细胞的活性（降低光毒性，减少荧光淬灭）4.长时间运行仪器各方面的稳定性植物显微成像的困难1.自发荧光强烈2.组织样品多样且很多比较致密3.不方便做切片植物活细胞显微成像的优势1.大部分时候无需控温以及CO22.比较容易得到有活性的组织DeltaVision Elite在植物领域的应用实例活细胞延时观察Four-dimensional imaging of centromeres during mitosis(中科院植物生理生态研究所中科院植物生理生态研究所)(Scale bar=5 m)7 sections0.5 m intervals7 sections0.5 m intervals45 secondsUp to 24 hoursThe actin cytoskeleton undergoes characteristic changes during pollen germination 单击此处编辑母版副标题样式北京大学生科院苏都莫日根课题组花花粉双受精粉双受精全内放射和宽场成像Image courtesy of Danny Nunez,Danuser Lab,Harvard Medical SchoolTIRFWidefield
