Parallelized Imaging and Automated Analysis of Zebrafish Assays with a Gigapixel Microscope

意义：高通量光学显微镜目前正在改变遗传学、药物发现等研究领域 和神经科学。大型光学分析现在通常使用数千张高分辨率图像来提供关键的见解 进入人体，我们的大脑和影响我们的疾病。今天的光学显微镜及其相关图像 然而，处理软件仍远未达到理想状态。目前的显微镜不能形成细胞级分辨率的图像 在一个超过几平方厘米的面积上，这从根本上限制了我们监测 生命系统。对于筛选许多活体模型生物，如斑马鱼，这一限制阻止了当前的设置 以细胞分辨率同时观察多个自由移动的生物，从而进行高分辨率的活体筛选 具有挑战性和耗时的实验。这也导致了缺乏有效的图像处理软件来处理高分辨率图像。 分辨率生物体图像分析。建议：在成功的一期和二期项目过程中，Ramona Optics 已经开发出一种新的“微摄像机阵列显微镜”(MCAM)，它通过使用 每帧高达1千兆像素，可以近细胞级别的细节解析数百个自由游泳的生物(5 微米/像素)。在这项计划中，Ramona Optics将生产一套充分利用MCAM的软件 信息丰富的录音。该软件将自动跟踪和测量关键的形态和荧光特征 在一个满满的井盘内同时跨越所有斑马鱼，以取代目前繁琐和耗时的任务。我们的MCAM 它的新软件将改变目前依赖于活体生物筛选的毒理学和药理学研究，通过 与目前的方法相比，在更少的时间内产生更多的实验见解，并具有更高的准确性和重复性。 SA1：用于并行幼虫跟踪和视频注册的MCAM软件：Ramona Optics将生产 用于跟踪、裁剪和注册MCAM FOV中的所有幼虫的Python软件，以生成以幼虫为中心的视频 随后进行了形态分析。我们将演示注册数百条幼虫图像的能力 同时(&lt；0.1%错误率)将保存的数据减少到原来的1/40，并生成标准化的每个生物体的数据集。 SA2：形态终点的自动标注：与俄勒冈州立大学坦盖实验室合作 大学和北卡罗来纳州州立大学的约德实验室，我们将创建算法来自动计算10条斑马鱼幼体 形态端点，例如凝视方向、胸鳍位置和身体曲率，我们将在一系列 毒理学滴定实验(在实验室中进行验证)，以证明至少比目前的筛查方法快100倍。 SA3：自动明场视频分析：然后我们将扩展我们的自动形态分析软件，以 明亮的视场视频，提供与时间相关的每个生物体的统计分析。我们将通过跟踪眼睛来展示这款软件 同时对96只幼虫进行角度检查，以显示作为毒物筛查新终点的视力(坦盖实验室)。 SA4：实现高通量荧光视频分析：我们将扩展我们的并行软件以监控 在96孔板内的所有幼虫中定位荧光，然后在免疫毒理学实验(Yoder Lab)中进行测试。 这个IIB阶段项目的结果将是一个旗舰MCAM软件套件，可供所有MCAM用户使用。

Significance: High-throughput optical microscopy is currently transforming the research fields of genetics, drug discovery and neuroscience. Large-scale optical assays now routinely use thousands of high-resolution images to offer critical insights into the human body, our brain and the diseases that affect us. Today's optical microscopes and their associated image processing software, however, are still far from ideal. Current microscopes cannot form images with cellular-scale resolution over an area larger than a few square centimeters, which fundamentally limits our ability to monitor the detailed movements of living systems. For screening many in vivo model organisms, such as zebrafish, this limitation prevents current setups from simultaneously observing multiple freely moving organisms at cellular resolution, which makes high-resolution in vivo screening experiments challenging and time-consuming. It has also led to a scarcity of effective image processing software for high- resolution organism image analysis. Proposal: Over the course of successful Phase I and Phase II projects, Ramona Optics has developed a new “micro-camera array microscope” (MCAM) that overcomes the above limitations by capturing video with up to 1 gigapixel per frame, which can resolve hundreds of freely-swimming organisms at near-cellular-level detail (5 µm/pixel). In this proposal, Ramona Optics will produce a suite of software that takes full advantage of the MCAM's information-rich recordings. This software will automatically track and measure key morphological and fluorescence features across all zebrafish within a full well plate, simultaneously, to replace currently tedious and time-consuming tasks. Our MCAM and its new software will transform current toxicology and pharmacology research that relies on in vivo organism screening, by producing more experimental insights in less time and with higher accuracy and repeatability than current methods. SA1: MCAM software for parallelized larval tracking and video registration: Ramona Optics will produce Python software to track, crop and register all larvae within the MCAM FOV to produce per-larvae centered video for subsequent morphological analysis. We will demonstrate the ability to register video of hundreds of larvae imaged simultaneously (<0.1% error rate) to reduce saved data by 40X and produce standardized per-organism datasets. SA2: Automated annotation of morphological endpoints: Working with the Tanguay Lab at Oregon State University and the Yoder Lab at NC State University, we will create algorithms to automatically compute 10 larval zebrafish morphological endpoints, such as gaze direction, pectoral fin position, and body curvature, which we will verify in a series of toxicology titration experiments (validated across labs) to demonstrate at least 100X speed-up to current screening methods. SA3: Automated bright-field video analysis: We will then extend our automated morphology analysis software to bright-field video to provide a time-dependent per-organism statistical analysis. We will showcase this software by tracking eye angle across 96 larvae simultaneously to demonstrate visual acuity as a new endpoint in toxicology screens (Tanguay Lab). SA4: Enabling high-throughput fluorescence video analysis: We will extend our parallelized software to monitor localized fluorescence across all larvae within a 96 well-plate, and then test it in an immunotoxicology experiment (Yoder Lab). The outcome of this Phase IIB project will be a flagship MCAM software suite ready for deployment to all MCAM users.