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

摘要 该项目的目标是为生物医学界带来一种新的光学成像技术， 建立了一个简单的，通用的和强大的替代共聚焦显微镜。我们的技术叫做HiLo 这是我们实验室在2008年夏天发明的。HiLo成像允许类似共焦的散焦 荧光背景抑制，而不使用共焦扫描机制。简而言之，它要求 采集两幅图像，一幅采用结构化照明，另一幅采用标准均匀照明， 照明。HiLo成像适用于荧光和非荧光样品。因为它是基于 传统的宽视场成像，它可以很容易地适应标准显微镜。此外，由于 仅需要两个图像，它可以以很高的采集速率操作，很容易超过视频速率（以 证明）。 该项目主要集中在仪器开发上。已经建立了基本的 HiLo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HiLo成像的多功能性 及其广泛的适用性，我们计划确认HiLo成像的有效性， 共焦显微术是不切实际或不可能的。具体来说，我们计划建立一个HiLo显微内镜， 和一个HiLo显微镜前者将用于在大鼠结肠中进行活体共焦成像， 与波士顿大学医学院胃肠病学的Satish Singh博士小组合作 目的是证明HiLo显微内镜用于光学活检的有效性， 癌症诊断我们的第二个设备，HiLo宏观，将利用HiLo成像的多功能性， 提供了共聚焦显微镜不能提供的，即视频速率，超过一个 非常宽的视场（约1厘米）和长的工作距离（几厘米）。这一应用 宏观将在小动物的分子成像，我们建议证明其有效性 与Rakesh Jain博士合作， 马萨诸塞州总医院放射肿瘤科的一个小组。 HiLo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， 它将成为生物医学成像的常规仪器。

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.