Integrated FMT approaches for biomolecular measurements

描述（由申请人提供）：在过去几年中，体内荧光成像已经取得了相当大的进展。在过去三年的资助中，（2006-2009）我们介绍了许多关于整个动物荧光断层成像（FMT）的进展：1）我们已经开发了理论和技术框架来执行非流体，非纤维系统FMT（“自由空间成像”），2）我们已经设计了在光学不均匀的漫射介质中以高保真度成像的算法，3）我们已经实现了快速反演方法，其允许在数秒-数分钟内重建大数据集，4）我们已经开发了结合计算机断层扫描（CT）或磁共振成像（MRI）的混合成像方法，5）我们已经构建了原型混合CT-FMT成像系统，以及6）我们已经开发了多通道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（上转换纳米粒子（UNP）和远红荧光蛋白，RFP）可能会产生相当大的影响。上转换纳米颗粒有望通过将照明转移到980 nm来基本上消除背景自发荧光。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的报告者的全部力量，需要新的重建算法，实验装置和严格的验证对公认的黄金标准。因此，该提案的目标是使当前的FMT仪器和算法适应新的报告基因，在模型中严格验证它们，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）系统和严格比较不同RFP肿瘤细胞系的体内成像，B）迭代地调整重建算法RFP的基础上，上述组织测量和c）使用优化的方法来定量体内细胞质量。在第二个目标（UNP成像）中，我们将调整和优化FMT-CT重建算法，用于体内UNP的检测，并基于UNP平台开发靶向组合PET-FMT试剂。在本次重新提交中，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成像的手稿，以及c）进一步澄清了上次审查期间提出的次要问题。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。 公共卫生相关性：这项研究的重点是开发下一代荧光技术，用于全身体内成像。在拟议的研究中，我们将实施两项最近发现的技术进步（红移荧光蛋白和新型荧光纳米材料），以提高体内断层荧光成像的定量准确性、检测灵敏度和特异性。

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.