The development of hybrid widefield imaging for out-of-focus background rejection

摘要 该项目的目标是将一种新的光学成像技术带到生物医学界，以及 将其确立为一种简单、通用和强大的替代共聚焦显微镜的方法。我们的技术叫做希洛 成像技术，是我们实验室在2008年夏天发明的。HELO成像允许像共焦一样的离焦 在不使用共聚焦扫描机制的情况下，荧光背景抑制。简而言之，它需要 采集两幅图像，一幅采用结构照明，另一幅采用标准均匀照明 照明。HELO成像适用于荧光和非荧光样品。因为它是基于 传统的广域成像，它可以很容易地改装到标准显微镜。而且，因为它 只需要两个图像，它可以在非常高的采集率下操作，轻松超过视频速率(到 被演示)。 这个项目主要集中在仪器开发上。已经建立了基本的 根据HALO显微镜的原理，我们计划通过实验量化关键性能参数，包括 轴向分辨率、信噪比和深度穿透。演示HELO成像的多功能性 和其广泛的适用性，我们计划确认HELO成像在以下情况下的有效性 共聚焦显微镜是不切实际的，甚至是不可能的。具体地说，我们计划建造一台Hilo内窥镜 和一台希洛的显微镜。前者将用于在大鼠结肠进行活体内共聚焦样成像，在 与波士顿大学医学院胃肠病学萨蒂什·辛格博士团队合作 部门，目的是展示HELO内窥镜在光学活组织检查和 癌症诊断。我们的第二个设备，希洛显微镜，将通过以下方式利用希洛成像的多功能性 提供共聚焦显微镜所不能提供的功能，即在 视场非常宽(大约1厘米)，工作距离很长(几厘米)。这一点的应用 宏观将在小动物的分子成像中，我们建议演示其有效性 与Rakesh Jain博士合作，在小鼠肿瘤模型中表征异常血管系统 小组在马萨诸塞州总医院放射肿瘤科。 HELO成像是一项新技术。我们的目标是牢固确立这项技术的优势，以便 它将成为生物医学成像中的常规仪器。

ABSTRACT The goal of this project is to bring a new optical imaging technology to the biomedical community, and establish it as a simple, versatile and robust alternative to confocal microscopy. Our technique is called HiLo imaging, and was invented by our lab in the summer of 2008. HiLo imaging allows confocal-like out-of-focus fluorescence background rejection without the use of a confocal scanning mechanism. In brief, it requires the acquisition of two images, one with structured illumination and another with standard uniform illumination. HiLo imaging works with both fluorescent and non-fluorescent samples. Since it is based on conventional widefield imaging, it can readily be adapted to standard microscopes. Moreover, since it requires only two images, it can be operated at very high acquisition rates that easily surpass video rate (to be demonstrated). This project is focused mostly on instrumentation development. Having already established the basic principles of HiLo microscopy, we plan to experimentally quantify key performance parameters, including axial resolution, signal to noise ratio and depth penetration. To demonstrate the versatility of HiLo imaging and its broad range of applicability, we plan to confirm the effectiveness of HiLo imaging in situations where confocal microscopy is impractical or impossible. Specifically, we plan to build both a HiLo endomicroscope and a HiLo macroscope. The former will be used to perform intravital confocal-like imaging in rat colons, in collaboration with the Dr. Satish Singh group at the Boston University School of Medicine Gastroenterology Department, with the goal of demonstrating the effectiveness of HiLo endomicroscopy for optical biopsy and cancer diagnosis. Our second device, a HiLo macroscope, will exploit the versatility of HiLo imaging by providing what confocal microscopy cannot, namely video-rate, out-of-focus background rejection over a very wide field of view (about 1cm) and with a long working distance (several cms). The applications of this macroscope will be in molecular imaging of small animals, and we propose to demonstrate its effectiveness at characterizing abnormal vasculature in mouse tumor models, in collaboration with the Dr. Rakesh Jain group at the Massachusetts General Hospital Department of Radiation Oncology. HiLo imaging is a new technology. Our goal is to firmly establish the advantages of this technology so that it will become routine instrumentation in biomedical imaging.