Fluorender: An Imaging Tool for Visualization and Analysis of Confocal Data as Ap

描述(申请人提供)：斑马鱼作为脊椎动物系统在分析体内细胞和组织结构和发育方面具有独特的优势。体外受精和透明的幼虫允许对活的、发育中的动物的细胞进行成像。新的基因技术可以敲除大多数斑马鱼基因，因此开发高通量技术来分析突变表型(“表型组计划”)变得至关重要。对于所有这些实验，共焦显微镜是一个必不可少的工具，因此开发软件来分析3D和4D(随时间变化的3D)共焦数据并快速得出与生物学相关的定性和定量结论同样至关重要。图像分析需要五个步骤：预处理、配准、可视化、分割和量化。我们已经开发了一个可下载的共焦分析应用程序--FluoRender，并针对可视化对其进行了优化，提供了相对于现有商业应用程序的显著优势。目前的提案将通过添加其他四个步骤使FluoRender成为一个功能齐全的包。我们的目标是使生物学家能够轻松分析3D和4D共焦数据，识别和测量感兴趣的特征，并允许快速重复分析多个样本。对于像分段这样受益于自动化的功能，我们提供用户验证和编辑，强调高精度而不是纯粹的速度。我们将重点研究三个具体问题：1)3D图像拼接，4D漂移去除；2)共焦数据的3D分割；3)共焦数据中的4D跟踪。马赛克技术可以扫描比显微镜视野更大的标本。时间推移实验得益于4D漂移的去除，因为成长中的胚胎可以改变形状，或者显微镜的焦点可以漂移。我们将开发三类对象的分割方法：核/细胞、轴突/树突和组织。我们将开发斑马鱼的半自动分割方法，以标记多个荧光团或光谱(Brain弓)。我们将开发方法，以便一旦细胞或组织最初被分割，就可以随着时间的推移进行跟踪，然后可视化地与原始数据共同注册，为解释它们的运动提供背景。改进后的FluoRender软件将提供一个自由分布的便携式套件，能够快速分析3D或4D共焦数据集。这将有助于分析野生型和突变胚胎的细胞运动、神经元电路和组织发育。鉴于斑马鱼作为人类疾病模型的相关性越来越大，拟议的分析软件有望有助于我们了解许多不同的发育、神经生物学和代谢性疾病。 公共卫生相关性：斑马鱼正被广泛用作人类发育、神经和生理疾病的模型。这个模型系统的一个重要优势是能够使用共聚焦显微镜和相关技术拍摄三维(3D)和四维(3D Over Time，或4D)图像，显示活斑马鱼胚胎和幼体的3D背景下细胞和器官的位置和结构。这项提议将开发新的3D/4D图像分析软件，有助于利用这些强大的成像技术来详细了解神经系统或器官结构和发育中可能发生的缺陷，并以了解人类疾病中的相关缺陷为长期目标。

DESCRIPTION (provided by applicant): The zebrafish offers unique advantages as a vertebrate system to analyze cell and tissue structure and development in vivo. External fertilization and transparent larvae allow imaging of cells in live, developing animals. New genetic techniques can knock out most zebrafish genes, making it critical to develop high- throughput techniques to analyze mutant phenotypes (the "phenome project"). For all these experiments, confocal microscopy is an essential tool, making it equally critical to develop software to analyze 3D and 4D (3D over time) confocal data and quickly derive biologically-relevant conclusions, both qualitative and quantitative. Image analysis requires five steps: preprocessing, registration, visualization, segmentation, and quantitation. We have already developed a downloadable confocal analysis application, FluoRender, and optimized it for visualization, providing significant advantages over existing commercial applications. The present proposal would make FluoRender a full-featured package by adding the other four steps. Our goal is to enable the biologist to easily analyze 3D and 4D confocal data, identifying and measuring features of interest, and allowing rapid repetitive analysis of multiple samples. For features like segmentation that benefit from automation, we provide user validation and editing, emphasizing high accuracy rather than pure speed. We will focus on three specific problems: 1) 3D image mosaicking, 4D drift removal; 2) 3D segmentation of confocal data; and 3) 4D tracking in confocal data. Mosaicking allows scanning of specimens larger than a microscope's field of view. Timelapse experiments benefit from 4D drift removal, since the growing embryo can change shape or the microscope's focus can drift. We will develop segmentation methods for objects from three classes: nuclei/cells, axons/dendrites, and tissues. We will develop semi-automatic segmentation methods for zebrafish labeled with multiple fluorophores, or spectrally (Brainbow). We will develop methods so that once cells or tissues are initially segmented, they can be tracked over time, then visualized co-registered with the raw data, providing context for interpreting their motion. The improved FluoRender software will provide a freely-distributed, portable suite that enables rapid analysis of 3D or 4D confocal datasets. This will aid in analyzing cell movements, neuronal circuitry, and tissue development in wildtype and mutant embryos. Given the growing relevance of zebrafish as a human disease model, the proposed analysis software can be expected to benefit our understanding of many different developmental, neurobiological, and metabolic diseases. PUBLIC HEALTH RELEVANCE: The zebrafish is becoming widely used as a model of developmental, neurological, and physiological diseases in humans. An important strength of this model system is the ability to use confocal microscopy and related techniques to take three-dimensional (3D) and four-dimensional (3D over time, or 4D) images, showing the positions and structures of cells and organs in the 3D context of living zebrafish embryos and larvae. This proposal would develop new 3D/4D image analysis software that would help to take advantage of these powerful imaging techniques to understand in detail the defects that can occur in nervous system or organ structure and development, with the long-term goal of understanding related defects in human disease.