Small Animal Tomography System for Green Fluorescent Protein Imaging

描述(由申请人提供)：这项提案的总体目标是开发用于小动物绿色荧光蛋白(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，小动物成像已经变得相当重要。对动物的疾病进展和治疗效果进行成像具有许多科学和经济优势，因为可以大幅减少在不同疾病阶段牺牲动物以及进行尸检和组织病理学研究。光学技术已被证明在应用于小动物成像时非常有价值，因为大量的光学标记可以在细胞和分子水平上靶向和可视化各种与疾病相关的过程。然而，到目前为止，大多数光学成像研究只探索了整个动物表面的成像，而没有层析重建。只有几个小组，包括我们的团队，公布了第一个断层扫描结果，揭示了三维荧光探针在小动物中的分布。在这些情况下，图像重建完全基于光在组织中传播的扩散模型，这是更普遍适用的传输模型的近似值。这限制了这种编码在涉及近红外发射的荧光团的研究中的应用，在近红外中，组织的吸收比可见光谱中的吸收要小。此外，只使用调制频率小于150 MHz的稳态仪器或频域设备。尽管前景看好，但这些系统仍然存在吸收和散射效应之间的串扰，空间分辨率有限，荧光吸收和寿命测定的准确性有限。将光学层析成像方法扩展到更短的波长是非常可取的，这将提供一种在可见光谱中发射光的绿色荧光蛋白质的成像手段。使用GFP及其衍生物，现在可以可视化任何细胞、组织或物种中的几乎任何感兴趣的蛋白质。研究人员致力于生物学各个层面的研究，如单分子动力学、细胞内蛋白质运输、细胞器动力学以及发育、癌症进展和其他疾病过程中的细胞和组织行为，目前主要利用GFP进行体外研究。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范围的研究领域产生重大影响。这一建议的主要假设是，通过使用基于辐射传输方程(也称为传输方程)的频域光传输模型的成像系统，可以克服现有光学层析成像仪器的局限性。与允许调制频率高于当前可用的150 MHz的仪器相结合，这将导致更好的空间分辨率，并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发荧光而产生的不良背景信号。这项提议的总体目标是开发新的光学方法，用于小动物的体积成像，用于研究，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.