High Resolution Multiplexed Fluorescence Tomography

描述（由申请人提供）：小动物荧光断层扫描已成为强大的生物医学研究工具，因为它允许在体内未受干扰的环境中对分子靶向荧光探针和红移荧光蛋白进行三维成像。尽管近年来在仪器和图像重建算法方面取得了重大进展，但荧光断层成像技术仍远未优化。两个关键限制是由于生物组织中的高度光散射导致的相对差的图像分辨率（限于1或2 mm），以及不能同时执行许多荧光靶的高通量成像（即“多路复用”），这主要是由于常见荧光团的光谱重叠、长的数据采集时间和组织自发荧光。 在这个项目中，我们将开发一种高度新颖的荧光断层扫描仪，它将使用一些独特的设计元素来解决这些关键限制，包括; i）通过动物对最早传输的并且因此最少散射的光子进行高速时间选通光子计数检测，从而允许成像分辨率接近250 μ m而不损失灵敏度或准确度，ii）每个轴向切片在约1分钟内快速获取高光谱和荧光寿命数据，允许至少五个并发荧光团的鲁棒去混合和组织自发荧光的拒绝，iii）用于激发红色和近红外区域中的几乎任何荧光团的高速脉冲超连续谱光源。该系统将在类似于X射线计算机断层扫描的仪器配置中以360度的连续轴向切片对小鼠进行成像。 我们将证明，扫描仪是能够高分辨率的多重成像，首先在复杂的荧光光学幻影与模拟背景自体荧光，其次在裸鼠与多重标记的人胶质瘤（Gli-36和GBM 8）肿瘤异种移植。我们预计该系统将在生物医学研究的许多领域中有应用，包括研究疾病的发展和对活动物非侵入性新疗法的反应。 公共卫生相关性：本项目的目标是开发一种高度新颖的荧光断层成像仪，用于在体内对整个动物中的分子探针和红移荧光蛋白进行高分辨率的多重成像。扫描仪的独特设计将允许在类似于X射线计算机断层扫描的光学仪器配置中快速、同时采集高光谱和时间数据集。新的图像重建算法将允许强大的去混合和可视化的至少五个荧光结构的分辨率接近250 5米，提供前所未有的小动物成像能力。我们预计该扫描仪将在生物医学研究中有许多应用，包括研究疾病的发展和对体内新疗法的反应。

DESCRIPTION (provided by applicant): Small animal fluorescence tomography has emerged as powerful biomedical research tool since it allows three-dimensional imaging of molecularly-targeted fluorescent probes and red-shifted fluorescent proteins in unperturbed environments in vivo. Despite significant advances in instrumentation and image reconstruction algorithms in recent years, fluorescence tomographic imaging technology remains far from optimized. Two critical limitations are the relatively poor image resolution (limited to 1 or 2 mm) due to the high degree of light scatter in biological tissues, and an inability to perform high-throughput imaging of many fluorescent targets simultaneously (i.e. "multiplexing"), largely due to the spectral overlap of common fluorophores, long data acquisition times and tissue autofluorescence. In this project, we will develop a highly novel fluorescence tomographic scanner that will address these critical limitations using a number of unique design elements including; i) high- speed time-gated photon counting detection of the earliest-transmitted and therefore least scattered photons through animals, thereby allowing imaging resolution approaching 250 5m without loss of sensitivity or accuracy, ii) fast acquisition of both hyperspectral and fluorescence lifetime data in about 1 minute per axial slice, allowing robust de-mixing of at least five concurrent fluorophores and rejection of tissue autofluorescence, iii) a high speed, pulsed supercontinuum light source for excitation of virtually any fluorophore in the red and near-infrared region. The system will image mice in sequential axial slices over 360 degrees in an instrument configuration analogous to X-ray Computed Tomography. We will demonstrate that the scanner is capable of high-resolution multiplexed imaging, first in complex fluorescent optical phantoms with simulated background autofluorescence, and secondly in nude mice with multiply-labeled human glioma (Gli-36 and GBM8) tumor xenografts. We anticipate that the system will have applications in many areas of biomedical research including studying disease development and response to novel therapeutics non- invasively in live animals. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a highly novel fluorescence tomographic imager for high- resolution multiplexed imaging of molecular probes and red-shifted fluorescent proteins in whole animals in vivo. The unique design of the scanner will allow rapid, concurrent acquisition of hyperspectral and temporal data sets in an optical instrument configuration analogous to X-ray Computed Tomography. Novel image reconstruction algorithms will allow robust de-mixing and visualization of at least five fluorescent constructs with a resolution close to 250 5m, offering unprecedented small animal imaging capabilities. We anticipate that the scanner will have many applications in biomedical research including studying disease development and response to novel therapeutics in vivo.