Small Animal Tomography System for Green Fluorescent Protein Imaging

描述（由申请人提供）：本提案的总体目标是开发用于小动物体内绿色荧光蛋白（GFP）断层成像的图像重建方案和仪器。近年来，随着越来越多的人类疾病动物模型的出现，小动物成像变得相当重要。动物疾病进展和治疗效果的成像具有许多科学和经济优势，因为可以大幅减少在各种疾病阶段处死动物并进行尸检和组织病理学研究。光学技术已被证明是非常有价值的，当应用于小动物成像，因为丰富的光学标记物，可以靶向和可视化各种疾病相关的过程中的细胞和分子水平。然而，迄今为止，大多数光学成像研究仅探索了整个动物表面成像，而没有断层重建。只有少数几个小组，包括我们的团队，提出了第一个断层扫描的结果，揭示了三维荧光探针分布在小动物。在这些情况下，图像重建仅基于光在组织中传播的扩散模型，该扩散模型是更普遍适用的传输模型的近似。这限制了此类代码在涉及近红外荧光团的研究中的应用，其中组织吸收小于可见光谱。此外，只有稳态仪器或调制频率小于150 MHz的频域设备。虽然有希望，但这些系统仍然受到吸收和散射效应之间的串扰、有限的空间分辨率以及荧光吸收和寿命测定的有限精度的影响。非常希望将光学层析成像方法扩展到更短的波长，这将提供对发射可见光谱中的光的绿色荧光蛋白成像的手段。使用GFP及其衍生物，现在可以可视化任何细胞，组织或物种中几乎任何感兴趣的蛋白质。研究人员在生物学的各个层面，如单分子动力学，细胞内的蛋白质运输，细胞器动力学，细胞和组织行为在发展，癌症进展和其他疾病，现在利用GFP主要是在体外研究。用于体内GFP成像的光学层析成像系统的可用性将对这一大领域的研究产生重大影响。该提案的主要假设是，与现有的光学层析成像仪器相关的限制可以通过使用基于辐射传输方程（也称为“传输方程”）的频域光传播模型的成像系统来克服。与允许调制频率高于目前可用的150 MHz的仪器相结合，这将导致更好的空间分辨率和减少散射和吸收效应之间的串扰。此外，使用多波长和多频率成像系统提供了独特的机会来减少由于自体荧光引起的不期望的背景信号。该提案的总体目标是开发用于研究的小动物体积成像的新光学方法，以更好地了解和治疗多种疾病。例如，成像系统可以帮助可视化肿瘤的位置和生长。如果成功实施，新的成像系统将为癌症等各种疾病如何传播和影响身体提供新的见解。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop image reconstruction schemes and instrumentation for tomographic imaging of green fluorescent proteins (GFP) in small animals. Small animal imaging has gained considerable importance in recent years as more and more animal models for human diseases have become available. Imaging of disease progression and effects of treatments in animals has many scientific and economical advantages, as sacrificing animals at various disease stages and performing necropsy and histopathological studies can be sharply reduced. Optical techniques have proven to be very valuable when applied to small animal imaging because of an abundance of optical markers that can target and visualize various disease related processes on the cellular and molecular level. However, to date most optical imaging studies have only explored whole animal surface imaging without tomographic reconstruction. Only a few groups including our team have presented first tomographic results that reveal three-dimensional fluorescent probe distribution in small animals. In these cases the image reconstructions were exclusively based on the diffusion model of light propagation in tissue, which is an approximation to the more generally applicable transport model. This has limited the application of such codes to studies involving fluorophores that emit in the near-infrared, where tissue absorption is smaller than in the visible spectrum. Furthermore, only steady-state instrumentation or frequency-domain devices with modulation frequencies smaller than 150 MHz were employed. While promising, these systems still suffer from cross-talk between absorption and scattering effects, limited spatial resolution, and limited accuracy in fluorescence absorption and lifetime determination. It is highly desirable to extend optical tomographic methods to shorter wavelengths, which would provide a means to image green fluorescence proteins that emit light in the visible spectrum. Using GFP and its derivatives, it is now possible to visualize nearly any protein of interest in any cell, tissue, or species. Researchers working at all levels of biology, such as single-molecule dynamics, protein trafficking within cells, organelle dynamics, and cell and tissue behaviors during development, cancer progression, and other diseases, are nowadays making use of GFP mostly in vitro studies. The availability of an optical tomography system for in vivo GFP imaging would have a significant impact on this large area of research. Themain hypothesis of this proposal is that limitations related to existing instrumentation for optical tomography can be overcome by an imaging system that uses a frequency-domain light propagation model that is based on the equation of radiative transfer (also called "transport equation"). Combined with instrumentation that allows for modulation frequencies higher than the currently available 150MHz, this will lead to better spatial resolution and reduced cross-talk between scattering and absorption effects. Furthermore, using a multi-wavelength and multi-frequency imaging system offers unique opportunities to reduce undesirable background signal due to autofluorescence. The overall goal of this proposal is to develop novel optical methods for volumetric imaging of small animals that are used in research to better understand and treat numerous diseases. For example, the imaging system could help visualize tumor location and growth. If successfully implemented the new imaging system will provide new insights into how various diseases, such as cancer, spread and affect the body.