Proj 1: In Vivo Optical Molecular Imaging with Fluorescence Peptides

项目1：本研究计划的广泛的长期目标是开发一种集成光学 使用荧光肽作为探针以靶向癌前病变的存在的分子成像策略 （发育不良）组织。研制了宏观和微观荧光仪器 作为肽检测的补充成像方法。该方法可用于监测 在小动物模型中的生物标志物表达和在接受癌症治疗的人类患者中的癌症早期检测 筛选在本申请中，结肠腺瘤被用作异型增生的模型。具体目标是 1)利用噬菌体展示技术筛选出5个候选肽段， 新鲜切除的人类结肠粘膜标本，2）使用 双轴共聚焦架构，以可视化与肽结合相关的组织微架构，以及3） 在基因工程小鼠体内验证肽与发育异常的优先结合， 通过Cre-重组酶的体细胞活化和在经历常规筛选的人类受试者中的远端结肠 结肠镜检查选择肽作为探针进行开发是因为它们的高度多样性和小尺寸 可以提供具有深组织穿透的高亲和力结合，并且它们的免疫原性风险低， 毒性促进翻译用于临床。噬菌体展示是一种强大的组合技术， 提供了一种无偏倚的方法，用于鉴定表现出与异型增生的亲和力结合的独特肽， 粘膜是非常适合于适应与体内微环境相关的细微变化的。的 双轴共焦结构提供了一种可以实现亚细胞分辨率的仪器 工作距离，并克服了使用离轴荧光收集的组织散射的影响。 此外，该设计可以按比例缩小尺寸以实现内窥镜兼容性。直径5毫米的仪器 包装足够小以穿过治疗内窥镜中的6 mm直径器械通道。 高速扫描是用一个微小的MEMS（微机电系统）镜，收集 以足够的帧速率生成3D体积图像的光学部分。候选肽将是 局部应用于结肠腺瘤和周围正常粘膜，然后肉眼观察 荧光成像以定位肽结合增加的区域，用于随后的显微荧光 成像以评估发育异常的隐窝微结构内肽结合的空间分布。 公共卫生：拟议的研究将导致新型光学成像探头的开发， 可以在临床前模型中进行评估，也可以在实际应用中转化为临床的工具 早期发现中空器官癌症的筛查工具。

PROJECT 1: The broad, long-term objectives of this research plan are to develop an integrated optical molecular imaging strategy that uses fluorescence peptides as probes to target the presence of premalignant (dysplastic) tissue in vivo. Macroscopic and microscopic fluorescence instruments are developed as complementary imaging methods for peptide detection. This methodology can be used for monitoring biomarker expression in small animal models and for the early detection of cancer in human patients undergoing screening. In this application, the colonic adenoma is used as a model for dysplasia. The specific aims are to 1) identify 5 candidate peptides using techniques of phage display for biopanning against cultured cells and freshly excised specimens of human colonic mucosa, 2) develop new optical imaging instrumentation using the dual axes confocal architecture to visualize the tissue microarchitecture relevant to peptide binding, and 3) validate preferential peptide binding to dysplasia in vivo in a genetically engineered mouse that forms polyps in the distal colon by somatic activation of Cre-recombinase and in human subjects undergoing routine screening colonoscopy. Peptides are chosen for development as probes because their high diversity and small size can provide high affinity binding with deep tissue penetration, and their low risk of immunogenicity and toxicity facilitate translation for use in the clinic. Phage display is a powerful combinatorial technique that provides an unbiased approach for identifying unique peptides that exhibit affinity binding to dysplastic mucosa as is well suited to accommodate subtle changes associated with the in vivo microenvironment. The dual axes confocal architecture provides an instrument that can achieve sub-cellular resolution with long working distance and overcomes the effects of tissue scattering using off-axis fluorescence collection. Moreover, this design can be scaled down in size for endoscope compatibility. A 5 mm diameter instrument package is sufficiently small to pass through a 6 mm diameter instrument channel in a therapeutic endoscope. High speed scanning is performed with a tiny MEMS (micro-electro-mechanical systems) mirror that collects optical sections at a sufficient frame rate for generating 3D volumetric images. The candidate peptides will be topically applied to colonic adenomas and surrounding normal appearing mucosa followed by macroscopic fluorescence imaging to localize regions of increased peptide binding for subsequent microscopic fluorescence imaging to assess the spatial distribution of peptide binding within the dysplastic crypt microarchitecture. Public Health: The proposed studies will result in the development of novel optical imaging probes and instruments that can be evaluated in pre-clinical models as well as translated to the clinic as practical screening tool for the early detection of cancer in hollow organs.