An automated image analysis tool for quantitative studies of dynamic 3D-wound healing assays

一个有效和快速的伤口愈合过程对于保持皮肤的完整性是必不可少的，因此对一个功能正常的生物体至关重要。皮肤损伤的原因多种多样，包括外力、手术或疾病。最近，三维（3D）伤口愈合模型已经成功建立。这些能够揭示伤口愈合过程中不同细胞类型的复杂相互作用。然而，可视化和分析仅在组织学切片上进行，这仅揭示了对潜在动力学的有限见解。我将建立一种3d体外光学显微镜技术和一种基于活细胞成像和自动化分析例程的新颖可视化工作流程，以分析整个体外3d皮肤模型的动态行为。我们将实施最近在生物应用中重新发现的成像方法-光片荧光显微镜(LSFM) -用于延时成像。因此，将首次阐明体外3d皮肤模型上伤口愈合的基本机制和动态方面。目前的3d组织研究缺乏一种通用的软件工具来自动和全面地分析时间分辨图像数据。基于我们研究小组和合作伙伴的专业知识，我们提出了一种软件工具，用于整个体外3d皮肤模型和伤口愈合分析的单细胞水平上的自动和公正的图像分析。此外，所开发的算法不仅限于皮肤，而且可以很容易地适应和应用于其他组织工程实验。探索3d皮肤模型，该算法将允许基于定义良好的提取生物物理特征，以无偏倚的方式表征皮肤的健康状态。这些特征将随着时间的推移而被跟踪，从而允许在高度动态的伤口愈合过程中量化变化。最后，我们将开发的算法作为高通量筛选（HTS）分析的自动化和无偏基准工具，用于在体外实验中测试各种伤口愈合介质和伤口覆盖物的性能。该跨学科项目结合了伤口愈合实验的生物物理研究和生物信息学，旨在为实验人员开发一种复杂且用户友好的生物信息学工具。

An effective and fast wound healing process is essential for preserving the integrity of the skin and thus crucial for a functioning organism. Causes of skin damage are various including external physical force, surgery or diseases. Lately, 3-dimensional (3D) wound healing models have been successfully established. These are capable of revealing the complex interplay of different cell types during wound healing processes. However, visualization and analysis have only been performed on histological sections, which reveal only limited insights into the underlying dynamics.I will establish a 3D-in-vitro optical microscopy technique and a novel visualization workflow based on live-cell imaging and automated analysis routines to analyze the dynamic behavior of a whole in-vitro 3D-skin model. We will implement a recently for biological applications rediscovered imaging method - light sheet fluorescence microscopy (LSFM) - for time-lapse imaging. Thus, for the first time the fundamental underlying mechanism and dynamic aspects of wound healing demonstrated on an in-vitro 3D-skin model will be elucidated. Current 3D-tissue studies are lacking a common software tool for automated and comprehensive analysis of time resolved image data. Based on the expertise in our research group and of our collaboration partners we propose a software tool for automated and unbiased image analysis on a single cell level for whole in-vitro 3D-skin models and wound healing assays. Moreover, the developed algorithms are not limited to skin but can be easily adapted and applied to other tissue engineering experiments.Exploring the 3D-skin model the algorithm will allow for characterizing the healthy state of the skin in an unbiased manner based on well-defined extracted biophysical features. These features will be tracked over time and thus allow quantification of changes during the highly dynamical process of wound healing. Finally, we will establish the developed algorithms as an automated and unbiased benchmarking tool for high throughput screening (HTS) assays for testing the performance of various wound healing mediators and wound covers in in-vitro experiments.The proposed interdisciplinary project combines both biophysical studies on wound healing experiments and bioinformatics in order to develop a sophisticated and user-friendly bioinformatics tool for experimentalists.