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

描述(申请人提供)：本项目的目标是将一种新的光学成像技术带到生物医学界，并将其确立为一种简单、通用和健壮的替代共聚焦显微镜的方法。我们的技术被称为希洛成像，是我们实验室在2008年夏天发明的。HELO成像允许在不使用共焦扫描机制的情况下进行类似于共焦的离焦荧光背景排斥。简而言之，它需要采集两幅图像，一幅是结构照明，另一幅是标准均匀照明。HELO成像适用于荧光和非荧光样品。由于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。此外，由于它只需要两个图像，它可以在非常高的采集速率下操作，轻松超过视频速率(待演示)。这个项目主要集中在仪器开发上。在建立了希洛显微镜的基本原理后，我们计划对关键性能参数进行实验量化，包括轴向分辨率、信噪比和深度穿透。为了证明HELO成像的多功能性及其广泛的适用范围，我们计划确认HELO成像在共焦显微镜不实用或不可能的情况下的有效性。具体地说，我们计划建造一台HELO内窥镜和一台HELO宏观显微镜。前者将与波士顿大学医学院胃肠病学系的萨蒂什·辛格博士团队合作，在大鼠结肠内进行活体共聚焦成像，目的是展示HELO内窥镜在光学活检和癌症诊断方面的有效性。我们的第二个设备，HELO显微镜，将利用HELO成像的多功能性，提供共焦显微镜无法提供的功能，即视频速率，在非常宽的视场(约1厘米)和长工作距离(几厘米)上的离焦背景抑制。该显微镜的应用将应用于小动物的分子成像，我们建议与马萨诸塞州总医院放射肿瘤科的Rakesh Jain博士团队合作，展示其在表征小鼠肿瘤模型中异常血管系统方面的有效性。HELO成像是一项新技术。我们的目标是牢固地确立这项技术的优势，使其成为生物医学成像中的常规仪器。 公共健康相关性：项目叙述公共健康：我们已经发明了一种新的光学成像技术来拒绝焦外背景，这种技术简单、廉价和多功能。我们建议开发一种基于这种技术的内窥镜和宽视场显微镜，用于临床前小动物癌症模型的成像和诊断。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Public health: We have invented a new optical imaging technology to reject out-of- focus background that is simple, inexpensive and versatile. We propose to develop an endomicroscope and a widefield macroscope based on this technology for pre-clinical small-animal cancer-model imaging and diagnosis.