Integrated FMT approaches for biomolecular measurements

描述（由申请人提供）：体内荧光成像在过去几年中取得了相当大的进展。在过去三年的资助（2006-2009）中，我们介绍了全动物荧光层析成像（FMT）的一些进展：1)我们开发了执行非流体、非光纤系统FMT（“自由空间成像”）的理论和技术框架，2)我们设计了在光学异构、扩散介质中进行高保真成像的算法，3)我们实现了快速反演方法，允许在几秒到几分钟内重建大型数据集，4)我们开发了混合成像方法，包括计算机断层扫描（CT）或磁共振成像（MRI），5)构建了CT-FMT混合成像系统原型；6)开发了多通道FMT。有许多独特的机会进一步推进FMT成像并将其应用于重要的生物医学问题。特别是，最近发现的两个新的报告探针平台（上转换纳米颗粒（UNP）和远红色荧光蛋白（RFP））可能会产生相当大的影响。上转换纳米粒子有望通过将照明转移到980纳米，从根本上消除背景自身荧光。另一方面，RFP将允许量化任何感兴趣的蛋白质（甚至可能是途径，细胞过程和蛋白质-蛋白质相互作用）。为了充分利用这些新记者的全部力量，需要新的重建算法、实验设置和针对公认金标准的严格验证。因此，本提案的目标是使当前的FMT仪器和算法适应新的报告，在幻影中严格验证它们，并将它们应用于高度相关的疾病小鼠模型。在第一个目标（RFP成像）中，我们将解决三个子主题：a)系统而严格地比较不同的RFP肿瘤细胞系进行体内成像，b)基于上述组织测量迭代地适应RFP重建算法，c)使用优化的方法定量体内细胞质量。在第二个目标（UNP成像）中，我们将调整和优化FMT-CT重建算法以检测体内UNP，并基于UNP平台开发靶向PET-FMT联合药物。在这篇重新提交的论文中，我们a)获得了关于PET-FMT融合实验可行性的大量新数据，b)发表了另外8篇关于混合FMT和/或UNP成像的论文，c)进一步澄清了之前综述中提出的一些小问题。我们相信这些新方法将提供更高的灵敏度和准确性，简化实验程序，并允许与其他成像模式和生物数据集无缝集成。

DESCRIPTION (provided by applicant): In vivo fluorescence imaging has seen considerable progress over the last several years. During the previous 3 years of funding (2006-2009) we have introduced a number of advances to whole animal fluorescence tomographic (FMT) imaging: 1) we have developed theoretical and technology frameworks to perform non- fluid, non-fiber system FMT ("free-space imaging"), 2) we have designed algorithm to image with high fidelity in optically heterogeneous, diffuse media, 3) we have implemented fast inversion methods that allow reconstructions of large data sets in seconds-minutes, 4) we have developed hybrid imaging approaches incorporating computed tomography (CT) or magnetic resonance imaging (MRI), 5) we have constructed a prototype hybrid CT-FMT imaging system and 6) we have developed multichannel FMT. There are a number of unique opportunities to further advance FMT imaging and apply it to important biomedical questions. In particular, the recent discovery of two new reporter probe platforms (upconverting nanoparticles (UNP) and far red fluorescent proteins, RFP) are likely to have considerable impact. Upconverting nanoparticles promise to essentially eliminate background autofluorescence by shifting illumination to 980 nm. RFP on the other hand, will allow the quantization of any protein of interest (potentially even pathways, cellular processes and protein- protein interactions). In order to harness the full power of these newer reporters, new reconstruction algorithm, experimental set-ups and rigorous validation against accepted gold-standards are required. The goal of this proposal is therefore to adapt current FMT instrumentation and algorithm to the new reporters, rigorously validate them in phantoms and apply them to highly relevant mouse models of disease. In the first aim (RFP imaging), we will address three sub-topics: a) a systematic and rigorous comparison of different RFP tumor cell lines for in vivo imaging, b) iteratively adapting the reconstruction algorithm for RFP based on the above tissue measurements and c) using the optimized approach to quantitate cell mass in vivo. In a second aim (UNP imaging) we will adapt and optimize FMT-CT reconstruction algorithm for the detection of UNP in vivo and developing targeted combined PET-FMT agents based on the UNP platform. In this resubmission we have a) obtained extensive new data on the feasibility of PET-FMT fusion experiments, b) have published another 8 manuscripts on hybrid FMT and/or UNP imaging and c) have further clarified minor concerns raised during the previous review. We believe that these new approaches will offer vastly higher sensitivity and accuracy, simplify experimental procedures and allow a seamless integration with other imaging modalities and biological data sets. PUBLIC HEALTH RELEVANCE: This research focuses on the development of next generation fluorescence technologies for whole body in vivo imaging. In the proposed research we will implement two recently discovered technical advances (red shifted fluorescent proteins and newer types of fluorescent nanomaterials) to advance the quantitation accuracy, detection sensitivity and specificity of in vivo tomographic fluorescence imaging.