Widefield fluorescence macroscopy with out-of-focus blur rejection

描述（由申请人提供）：我们的目标是开发一种新型的成像“宏观镜”，其在厚组织中执行超深（几毫米）荧光成像，同时提供散焦背景抑制。我们所提出的方法为离焦模糊抑制是基于一种新的技术，称为动态散斑照明（DSI）显微镜，最近在我们的实验室发明的。我们的目标是建造一台便携式DSI宏观镜，并确定其在体内分子成像应用（例如癌症研究和诊断以及小动物成像和手术）中的潜力。DSI显微镜由对标准宽视场荧光显微镜的简单修改组成。用随机激光散斑图案而不是用灯进行样品照明。DSI显微镜的主要优点是，它提供了深度歧视和离焦模糊减少厚组织，而无需使用复杂的扫描机制。我们已经开发了DSI显微镜的完整理论，并且已经证明了亚微米分辨率成像与GFP标记神经元的共焦样背景排斥，在小鼠大脑中低至约100 μ m。我们建议开发一种新的仪器，执行更深的成像，但分辨率较低。我们的目标深度是几毫米，甚至一厘米。为了实现这一目标，我们建议显着重新设计我们的DSI显微镜，将一个长的工作距离远心物镜，近红外激光照明，和一个更敏感的CCD相机。我们还计划将联合收割机DSI与结构照明对比度和多光谱成像相结合（对于后者，我们将与剑桥研究与仪器公司合作）。与传统的市售宏观镜相比，我们的DSI宏观镜的定义优势在于，DSI提供了失焦背景抑制，能够显著更好地定位和可视化厚组织内的标记结构。我们的DSI原型的初始测试将在放射实验室提供的组织体模上进行。具有离焦模糊抑制的宽视场荧光宏观检查。公共卫生：我们建议开发一种简单而廉价的设备，用于小动物癌症模型成像，该设备可以进行具有离焦模糊抑制的超深荧光成像。我们的目标是建立一个新的癌症研究工具，最终可以在临床上实施。

DESCRIPTION (provided by applicant): Our aim is to develop of a new type of imaging "macroscope" that performs ultra-deep (several millimeters) fluorescence imaging in thick tissue while providing out-of-focus background rejection. Our proposed method for out-of-focus blur rejection is based on a novel technique called Dynamic Speckle Illumination (DSI) microscopy that was recently invented in our lab. Our goal is to build a portable DSI macroscope and establish its potential for in-vivo molecular-imaging applications such as cancer research and diagnosis, as well as small animal imaging and surgery. DSI microscopy consists of a simple modification to a standard widefield fluorescence microscope. Sample illumination is performed with random laser speckle patterns rather than with a lamp. The main advantage of DSI microscopy is that it provides depth discrimination and out-of-focus blur reduction in thick tissues without the use of a complicated scanning mechanism. We have developed the full theory of DSI microscopy and already demonstrated sub-micron-resolution imaging with confocal-like background rejection of GFP-labeled neurons, down to about 100¿m in mice brain. We propose to develop a new instrument that performs much deeper imaging but with lower resolution. Our targeted depth is several millimeters, down to perhaps a centimeter. To attain this goal we propose to significantly re-design of our DSI microscope to incorporate a long working-distance telecentric objective, near-infrared laser illumination, and a more sensitive CCD camera. We also plan to combine DSI with structured illumination contrast and multipsectral imaging (for the latter, we will work with Cambridge Research & Instrumentation). The defining advantage of our DSI macroscope compared to conventional commercially available macroscopes will be that DSI provides out-of-focus background rejection, enabling significantly better localization and visualization of labeled structures within thick tissue. Initial testing of our DSI prototype will be performed on tissue phantoms provided by a radiology lab. Widefield fluorescence macroscopy with out-of-focus blur rejection. Public health: We propose to develop a simple and inexpensive device that performs ultra-deep fluorescence imaging with out-of-focus blur rejection, for small animal cancer-model imaging. Our goal is to build a new tool for cancer research that can eventually be implemented in the clinic.