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3D Quantitative Fluorescent Speckle Microscopy

3D 定量荧光散斑显微镜

基本信息

批准号：
9752630
负责人：
Gaudenz Danuser
金额：
$ 40.5万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9752630
关键词：
3-Dimensional Actins Actomyosin Adopted Adoption Affect Automobile Driving Benchmarking Biochemical Biological Cell Adhesion Cell Surface Receptors Cell model Cell physiology Cell-Matrix Junction Cells Cellular Structures Cellular biology Clathrin Collaborations Color Communities Computers Computing Methodologies Coupled Cultured Cells Cytoskeleton Data Set Dedications Development Dimensions Engineering Environment Epithelial Cells Excision Eye Fluorescence Microscopy Fluorescent Probes Focal Adhesions Funding Gene Expression Generations Geometry Glass Goals Grant Image Imagery Imaging Techniques Imaging technology In Vitro Individual Infrastructure Intuition Label Life Light Macromolecular Complexes Maps Measures Mechanics Meiosis Memory Methods Microscopy Microtubules Mitotic Modeling Molecular Molecular Structure Monitor Morphogenesis Motion Nature Network-based Optics Organelles Organizational Models Pattern Physiological Plus End of the Microtubule Polymers Population Positioning Attribute Post-Translational Protein Processing Preparation Process Progress Reports Regulation Research Resolution Salmon Sampling Signal Transduction Slide Structure Subcellular structure Surface Techniques Technology Testing Texture Three-Dimensional Image Time Tissue Model Tissues Training Visual Work base cell assembly cell cortex cell motility coated pit computerized tools design experimental group experimental study extracellular flexibility high resolution imaging imaging capabilities imaging modality imaging study interest light microscopy live cell imaging macromolecular assembly microscopic imaging migration molecular assembly/self assembly molecular dynamics movie nanometer novel operation particle polarized cell prevent quantitative imaging reconstitution routine imaging single molecule spatiotemporal technological innovation tissue culture tool two-dimensional

项目摘要

ABSTRACT Fluorescent Speckle Microscopy (FSM) is an imaging mode to visualize and quantify the dynamics of macromolecular assemblies in living cells. It relies on vastly substoichiometric labeling of one or several components of the assembly of interest. When imaged by diffraction limited optics this labeling generates a random punctate texture that encodes in a statistical fashion transport, mechanical deformation, and molecular turnover of the assembly. As such, FSM is related to the super-resolution techniques STORM and PALM, which both rely also on random sampling of the molecular constituents of macromolecular assemblies. In STORM and PALM substochiometry in labeling is achieved by passive or active switching of a small set of fluorescent probes between a dark and a bright state. In contrast, in the original implementation FSM has relied on a population of permanently labeled subunits that dynamically incorporate in the assembly. In STORM and PALM, substochiometric labeling is exploited to sequentially collect the coordinates of individual subunits with nanometer precision, i.e. to acquire over time a super-resolution map of the molecular organization of an assembly. In FSM, substochiometric labeling is exploited to track in real-time subunit motion, addition and removal. Accordingly, the spatial resolution of FSM is still diffraction-limited; however, FSM offers information about the dynamics of a macromolecular assembly no other imaging modality provides. FSM has seen widespread applications in the research of cytoskeleton dynamics. Under the auspices of the present grant, my lab has developed the computational approaches required to make FSM a quantitative imaging technique (qFSM). In collaboration with several experimental groups as well as by developing FSM imaging capabilities in my own lab we have used qFSM technology to study the dynamics of the actin and microtubule cytoskeletons and associated molecular structures in cell morphogenesis, migration and division; and extended the method to the analysis of transient assembly of cell surface receptors in cellular signaling. Due to its rigid requirements for diffraction-limited imaging qFSM has been restricted, however, to live imaging of cells cultured on glass slides, which is entirely unphysiological. Capitalizing on the recent revolution in light-sheet imaging we propose here to take qFSM to the third dimension in order to apply its power for unveiling cytoskeleton dynamics in organotypic models of cells and tissues. This endeavor will require an iterative optimization of i) the design and implementation of multispectral light-sheet microscopy; ii) the flexible and simultaneous, substoichiometric labeling of multiple macromolecular assemblies, iii) the development of computational tools for tracking and interpretation of speckle dynamics in 3D time-lapse volumes. Specifically, in Aim 1, we will focus on molecular aggregates tracking microtubule plus ends and on clathrin-coated pits to develop fast 3D image acquisition and highly-sensitive 3D particle tracking methods with the goal of measuring the lifetime of macromolecular assemblies. In Aim 2, we will focus on the dynamics of the actomyosin cell cortex during cell polarization to develop robust dual-color speckle generation and optical-flow based computational methods with the goal of spatiotemporally mapping rates of molecular turnover and contraction in the cell cortical network. In Aim 3, we will focus on interactions between actomyosin cell cortex, components of cell adhesions, and the collagenous 3D microenvironment of cells to develop simultaneous 4- color image acquisition of speckle patterns and the computational tools for quantification and visualization of the coupled dynamics of macromolecular assemblies with the goal of testing the null hypothesis of a molecular clutch between cortical network and cell matrix adhesions in 3D. All tools will be engineered with an eye towards generalization, so that our technological innovations can be rapidly deployed to the community for the study of other dynamic cell structures.

摘要荧光散斑显微镜（FSM）是一种成像模式，用于可视化和量化活细胞中大分子组装的动力学。它依赖于大大低于化学计量的标记感兴趣组件的一个或多个组件。当通过衍射成像时有限的光学，这种标记产生随机的点状纹理，时尚运输，机械变形，和分子翻转的组装。因此，在本发明中， FSM与超分辨率技术STORM和PALM相关，两者都依赖于随机抽样的分子组成的大分子组件。在风暴和标记中的PALM亚化学计量是通过一小部分化学计量的被动或主动切换来实现的。荧光探针之间的黑暗和明亮的状态。相比之下，在最初的实现中， FSM依赖于一群永久标记的亚基，这些亚基动态地整合到大会。在STORM和PALM中，利用substochiometric标记来顺序地以纳米精度收集各个子单元的坐标，即随着时间的推移获取一个集合体分子组织的超分辨率图。在密克罗尼西亚联邦，利用标记来实时跟踪子单元运动、添加和移除。因此 FSM的空间分辨率仍然是衍射限制的;然而，FSM提供了关于大分子组装的动力学没有其他成像模式提供。FSM看到在细胞骨架动力学研究中有着广泛的应用。主持下目前，我的实验室已经开发出了使FSM成为一种定量成像技术（qFSM）。与几个实验小组合作，通过在我自己的实验室开发FSM成像功能，我们使用了qFSM技术研究肌动蛋白和微管细胞骨架及其相关分子的动力学，细胞形态发生，迁移和分裂的结构;并将该方法扩展到分析细胞信号传导中细胞表面受体的瞬时组装。由于其刚性然而，衍射限制成像qFSM的要求已被限制为实时成像在载玻片上培养的细胞，这是完全不符合生理的。利用最近的我们在这里建议将qFSM带到第三维，以便应用其力量来揭示细胞器官模型中的细胞骨架动力学，组织中这一努力将需要迭代优化i）设计和实现的多光谱光片显微镜; ii）灵活和同时，亚化学计量标记多个大分子组装体，iii）开发计算工具，跟踪和解释3D延时体积中的散斑动力学。具体而言，在目标1中，我们将重点关注跟踪微管加末端的分子聚集体和网格蛋白包被的开发快速3D图像采集和高灵敏度3D颗粒跟踪方法，测量大分子集合体寿命的目标。在目标2中，我们将重点关注在细胞极化过程中肌动球蛋白细胞皮质的动力学，以形成稳健的双色散斑生成和基于光流的计算方法，其目标是细胞皮质中分子周转和收缩的时空映射率网络在目标3中，我们将重点关注肌动球蛋白细胞皮质，细胞粘附，以及细胞的胶原3D微环境，以同时产生4- 彩色图像采集的散斑图案和计算工具的量化和可视化的耦合动力学的大分子组件的目标是测试 3D中皮质网络和细胞基质粘附之间的分子离合器的零假设。所有工具都将着眼于通用化，以便我们的技术创新可以迅速部署到社会上的其他动态细胞的研究结构.

项目成果

期刊论文数量（29）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Imaging assay to probe the role of telomere length shortening on telomere-gene interactions in single cells.

DOI：
10.1007/s00412-020-00747-4
发表时间：
2021-03
期刊：
Chromosoma
影响因子：
1.6
作者：
Zhang N;Li Y;Lai TP;Shay JW;Danuser G
通讯作者：
Danuser G

Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation.

DOI：
10.7554/elife.66151
发表时间：
2021-03-30
期刊：
eLife
影响因子：
7.7
作者：
Han SJ;Azarova EV;Whitewood AJ;Bachir A;Guttierrez E;Groisman A;Horwitz AR;Goult BT;Dean KM;Danuser G
通讯作者：
Danuser G

Dynamic macrophage "probing" is required for the efficient capture of phagocytic targets.

DOI：
10.1083/jcb.201007056
发表时间：
2010-12-13
期刊：
The Journal of cell biology
影响因子：
0
作者：
Flannagan RS;Harrison RE;Yip CM;Jaqaman K;Grinstein S
通讯作者：
Grinstein S

plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.

DOI：
10.1016/j.jsb.2011.07.009
发表时间：
2011-11
期刊：
JOURNAL OF STRUCTURAL BIOLOGY
影响因子：
3
作者：
Applegate, Kathryn T.;Besson, Sebastien;Matov, Alexandre;Bagonis, Maria H.;Jaqaman, Khuloud;Danuser, Gaudenz
通讯作者：
Danuser, Gaudenz

Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.