High Resolution Multiplexed Fluorescence Tomography

描述(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，因为它可以在体内不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，但荧光层析成像技术仍然远远没有得到优化。两个关键的限制是由于生物组织中的高度光散射而导致的图像分辨率相对较低(限于1或2 mm)，以及无法同时对许多荧光目标进行高通量成像(即多路传输)，这主要是由于普通荧光团的光谱重叠、较长的数据采集时间和组织自身荧光。在本项目中，我们将开发一种高度新颖的荧光断层扫描仪，该扫描仪将利用一些独特的设计元素来解决这些关键限制，包括：i)高速时间门控光子计数检测通过动物传输的最早，因此最少散射的光子，从而使成像分辨率接近250 5m而不会损失灵敏度或精度；ii)每个轴向切片约1分钟内快速获取高光谱和荧光寿命数据，从而实现至少五个并发荧光团的强劲分解和组织自发荧光的抑制；iii)高速脉冲超连续谱光源，用于激发几乎所有红色和近红外区域的荧光团。该系统将在一种类似于X射线计算机断层扫描的仪器配置中，以360度的顺序对小鼠进行轴向切片成像。我们将展示该扫描仪能够进行高分辨率多路成像，首先是在具有模拟背景自体荧光的复杂荧光光学模体中，然后是在具有多标记人脑胶质瘤(Gli-36和GBM8)肿瘤异种移植瘤的裸鼠身上。我们预计该系统将在生物医学研究的许多领域中得到应用，包括研究活体动物的疾病发展和对新疗法的非侵入性反应。 与公众健康相关：该项目的目标是开发一种高度新颖的荧光断层成像仪，用于在整个动物体内对分子探针和红移荧光蛋白进行高分辨率多路成像。该扫描仪独特的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至少五个分辨率接近250 5m的荧光结构的稳健分解和可视化，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，包括研究体内疾病的发展和对新疗法的反应。

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.