Collaborative Research: Development of Novel Two-photon Fluorescence Polymer Probes for High Resolution Deep Tissue Intravital Imaging

1403525/1403535凯文D. Belfield，University of Central Florida小松，Masanobu，Sanford-Burnham医学研究所新型双光子荧光聚合物探针用于高分辨率深部组织活体成像的开发重要性生物光子材料和过程正在迅速普及于许多新兴技术中，从电信到生物医学成像，这些技术对社会具有深远的影响。 体内成像，再加上高效的多功能纳米探针，有望以一种可能对社会福祉产生深远影响的方式对纳米医学和诊断领域产生变革性影响。用于深部组织成像的双光子荧光显微镜（2 PFM）是一种具有独特亚细胞分辨率的新兴技术。为了充分利用2 PFM，具有高双光子吸收（2 PA）截面和高荧光量子产率的荧光探针是至关重要的，同时是无毒的，不含重金属。纳米医学和诊断学的未来在于多功能纳米平台的开发，该多功能纳米平台将高度特异性靶向和高效成像功能联合收割机结合起来，促进时间和空间位点特异性成像，而不是在仔细控制的细胞培养中，而是在活组织中。 这个高度跨学科的建议旨在开发先进的聚合物为基础的探针在体内2 PFM血管生成成像。体内2 PFM可以提供实时、微创的肿瘤进展监测，肿瘤对抗癌治疗的反应，以及比目前已知的更详细的伤口愈合。该项目为先进材料和显微技术的跨学科研究、培训和教育提供了一个极好的平台。特别鼓励传统上在科学和工程领域代表性不足的学生参加这个项目。通过Belfield参与欧盟委员会的FP 7玛丽居里行动计划，将有强大的国际参与，支持跨学科培训，多样化，全球熟练，高技能的劳动力的发展。技术说明很少有努力已经报告的方法，以推动限制和提高对比度和质量的双光子荧光显微镜（2 PFM）在组织成像。在该提案中，我们建议通过控制多价性，整合多个高效的2 PA，近红外发射荧光团和靶向聚合物纳米平台中特定生物标志物的载体，显着提高2 PFM成像中的探针性能和靶向选择性。 由于纳米探针的小尺寸和生物相容性，这种方法有望通过增加亲合力和生物利用度来增强细胞相互作用。我们提出了一种聚合物纳米平台方法来制备定义明确的探针，提供高的局部荧光团浓度，以实现高对比度成像和同一聚合物链上的多个靶向部分，以提高亲和力，从而减少所需的剂量。预计这将提供从组织内更深处进行亚细胞分辨率的光学切片，并使其能够在三维中跟踪生物学上重要的事件，例如血管尖端细胞的发芽。肿瘤血管生成是一个复杂的生物学过程，只能在活体动物中进行研究。然而，在体内肿瘤血管生成的研究技术是严重缺乏。 我们将通过研究血管生成和血管细胞的生长和运动来推动体内2 PFM的极限，这些过程对许多疾病的发病机制很重要，同时帮助我们更好地了解肿瘤血管生成的生物学。

1403525/1403535Kevin D. Belfield, University of Central FloridaKomatsu, Masanobu, Sanford-Burnham Medical Research Institute Development of Novel Two-photon Fluorescence Polymer Probes for High Resolution Deep Tissue Intravital ImagingSignificanceBiophotonic materials and processes are fast becoming pervasive in many new and emerging technologies from telecommunications to biomedical imaging, technologies that have far reaching impact on society. In vivo imaging, coupled with efficient multifunctional nanoprobes, promises to transformationally impact the field of nanomedicine and diagnostics in a manner that may have profound effects on societal well being. Two-photon fluorescence microscopy (2PFM) for deep tissue imaging is a developing technology with unique subcellular resolution. To take full advantage of 2PFM, fluorescence probes with high two-photon absorption (2PA) cross-sections and high fluorescence quantum yields are critical all the while being nontoxic and heavy-metal free. The future of nanomedicine and diagnostics lies in the development of multifunctional nanoplatforms that combine both highly specific targeting and efficient imaging functionality, facilitating temporal and spatial site-specific imaging, not in carefully controlled cell culture but in vivo in living tissue. This highly interdisciplinary proposal aims to develop advanced polymer-based probes for in vivo 2PFM angiogenesis imaging. In vivo 2PFM may provide real-time, minimally invasive monitoring of tumor progression, a tumor's response to anti-cancer therapies, and wound healing in much greater detail than is currently known. This project constitutes an excellent platform for interdisciplinary research training and education in advanced materials and microscopy techniques. Students that are traditionally underrepresented in science and engineering will be especially encouraged to participate in this project. There will be strong international engagement through Belfield's participation in the European Commission's FP7 Marie Curie Actions program, supporting the development of an interdisciplinary-trained, diverse, globally adept, highly-skilled workforce.Technical DescriptionFew efforts have been reported on approaches to push the limits and improve the contrast and quality of two-photon fluorescence microscopy (2PFM) imaging in tissue. In this proposal, we propose to dramatically improve probe performance and targeting selectivity in 2PFM imaging through controlled multivalency, integrating multiple highly efficient 2PA, near-IR emitting fluorophores and vectors that target specific biomarkers in a polymeric nanoplatform. This approach is expected to enhance cellular interaction through increased avidity and bioavailability due to the small size and biocompatibility of the nanoprobe. We propose a polymeric nanoplatform approach to prepare well-defined probes that provide high local fluorophore concentration to achieve high contrast imaging and multiple targeting moieties on the same polymer chain to enhance avidity, hence reducing the dose needed. This is expected to provide optical sectioning with subcellular resolution from deeper within tissue and make it possible to track biologically important events, such as sprouting of vessel tip cells, in three dimensions. Tumor angiogenesis is a complex biological process that can only be studied in living animals. However, techniques for the study of tumor angiogenesis in vivo are acutely lacking. We will push the limits of in vivo 2PFM by studying angiogenesis and vascular cell growth and movement, processes important for the pathogenesis of a number of diseases, while helping us gain a better understanding of the biology of tumor angiogenesis.