An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images
An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images
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DOI：
10.3791/4233
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发表时间：
2012-08-01
影响因子：
1.2
通讯作者：
Bartlett, Selena E.
中科院分区：
文献类型：
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作者：
Haass-Koffler, Carolina L.;Naeemuddin, Mohammad;Bartlett, Selena E.
可用于测量荧光图像的最常见的软件分析工具是二维（2D）数据，这些数据依赖于手动设置来包含和排除数据点，以及计算机辅助模式识别来支持分析的解释和结果。能够测量从三维（3D）数据集构建的荧光图像变得越来越重要，以便能够捕获细胞动力学的复杂性并了解生物系统内细胞可塑性的基础。先进的显微镜仪器已经允许通过采集多光谱荧光图像和强大的分析软件来可视化3D荧光图像，该分析软件从共焦堆栈重建图像，然后提供所收集的2D图像的3D表示。先进的基于设计的体视学方法已经从原始的基于模型的体视学（1）的近似和假设发展而来，即使在复杂的组织切片（2）中也是如此。尽管在显微镜的这些科学进步，仍然需要一个自动化的分析方法，充分利用内在的3D数据，以允许分析和量化的复杂变化，细胞形态，蛋白质定位和受体trafficking.Current技术可用于量化荧光图像包括Meta-Morph（分子设备，桑尼维尔，CA）和图像J（NIH），提供手动分析。Imaris（Andor Technology，贝尔法斯特，北方爱尔兰）软件提供了MeasurementPro功能，该功能允许手动创建测量点，这些测量点可以放置在体积图像中或绘制在一系列2D切片上以创建3D对象。此方法适用于单击点测量，以测量两个对象之间的直线距离或创建包围感兴趣区域的多边形，但很难应用于复杂的蜂窝网络结构。Filament Tracer（Andor）允许自动检测3D神经元类神经元，然而，该模块已被开发用于测量定义的结构，例如神经元，其由树突，轴突和棘（树状结构）组成。该模块已被巧妙地用于对非神经元细胞进行形态学测量（3），然而，输出数据通过使用依赖于定义的细胞形状而不是无定形细胞模型的软件来提供扩展的细胞网络的信息。为了克服分析无定形细胞的问题，使软件更适合生物学应用，Imaris开发了Imaris Cell。这是Eidgenossische Technische Hochschule的一个科学项目，该项目旨在计算细胞和细胞器之间的关系。虽然该软件能够通过强制每个细胞一个细胞核并使用细胞膜来分割细胞来检测生物学约束，但它不能用于分析不连续的荧光数据，因为理想情况下它构建的细胞表面没有空隙空间。据我们所知，目前还没有开发出用户可修改的自动化方法，该方法可以从3D荧光图像中提供形态信息，从而实现未定义形状的细胞空间信息（图1）。我们已经开发了一个分析平台，该平台使用Imaris核心软件模块和与MATLAB（Mat Works，Inc.）接口的Imaris XT。这些工具允许对没有预定义形状和不一致荧光网络组件的细胞进行3D测量。此外，这种方法将允许在生物系统中具有扩展专业知识但不熟悉计算机应用的研究人员对细胞动力学中的形态变化进行量化。
The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology(1) even in complex tissue sections(2). Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking.Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells(3), however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenossische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1).We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.