Integrated FMT approaches for biomolecular measurements

描述(申请人提供)：活体荧光成像在过去几年中取得了长足的进步。在过去三年的资助期间(2006-2009)，我们在全动物荧光断层扫描(FMT)成像方面取得了许多进展：1)我们开发了执行非液体、非纤维系统FMT(“自由空间成像”)的理论和技术框架，2)我们设计了在光学不均匀、漫反射介质中高保真成像的算法，3)我们实现了快速反转方法，允许在几秒钟内重建大数据集，4)我们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)搭建了CT-FMT混合成像系统样机；6)研制了多通道FMT系统。有许多独特的机会来进一步推进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)和远红荧光蛋白，RFP)可能会产生相当大的影响。通过将光照转换到980 nm，上转换纳米颗粒有望从根本上消除背景自发荧光。另一方面，RFP将允许对任何感兴趣的蛋白质(甚至可能是通路、细胞过程和蛋白质-蛋白质相互作用)进行量化。为了充分利用这些较新的记者，需要新的重建算法、实验设置和针对公认的黄金标准的严格验证。因此，这项提议的目标是使当前的FMT仪器和算法适应新的记者，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)用于活体成像的不同RFP肿瘤细胞系的系统和严格的比较，b)基于上述组织测量迭代地采用RFP重建算法，以及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，我们将调整和优化用于体内UNP检测的FMT-CT重建算法，并基于UNP平台开发靶向的PET-FMT联合试剂。在这次重新提交中，我们a)获得了关于PET-FMT融合实验可行性的广泛新数据，b)发表了另外8篇关于混合FMT和/或UNP成像的手稿，以及c)进一步澄清了在前一次审查中提出的次要问题。我们相信，这些新的方法将提供更高的灵敏度和准确性，简化实验程序，并允许与其他成像方式和生物数据集的无缝集成。

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.