Novel Confocal Microscope for Molecular/Cellular Imaging

用于分子/细胞成像的新型共焦显微镜

• 批准号: 7105578
• 负责人: Thomas D Wang
• 金额: $ 12.57万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105578
• 关键词: Barretts esophagus; bioimaging /biomedical imaging; biomedical equipment development; biopsy; clinical biomedical equipment; clinical research; colon neoplasms; confocal scanning microscopy; diagnosis design /evaluation; endoscopy; fluorescence microscopy; green fluorescent proteins; histopathology; human subject; image enhancement; laboratory mouse; medulloblastoma; miniature biomedical equipment; mucosa; neoplasm /cancer diagnosis; neoplasm /cancer transplantation; preneoplastic state; tissue /cell culture

DESCRIPTION (provided by applicant): We would like to develop a new method of in vivo imaging using a miniaturized confocal microscope to study the biological processes of tumor development on the molecular and cellular level. This method has the potential to revolutionize cancer research. The long term objective of this project is to develop a technique to observe how tumors originate, progress, and invade. The behavior of cells will be monitored from images of sub-cellular constituents tagged with green or yellow fluorescence protein (GFP, YFP). A miniaturized confocal prototype has already been developed and its performance has been demonstrated with ex vivo images. We plan to improve this microscope with a new design using a dual-angle-axis architecture. This design offers improved resolution and working distance, which will facilitate in vivo imaging. We then plan to study two tumor models, medulloblastoma in a mouse model that has been associated with decreased expression of the ptc1 gene, and dysplasia in Barrett's esophagus and colonic adenomas. We will use these models to develop the new microscope. First, the performance of the tabletop dual-angle-axis prototype will be characterized by reflectance and fluorescence images of cultured cells and tissues sections expressing GFP/YFP and by biopsy specimens of Barrett's esophagus and colonic adenomas. Then, images will be collected from post-natal ptc1 knock-out mice expressing GFP/YFP to monitor angiogenesis, tumor invasion, and response to therapy. Next, the results of these studies will be used to develop the redesigned miniaturized microscope using Micro-Electro-Mechanical Systems (MEMS) fabrication technology. The miniaturized microscope will be used to study medulloblastoma in ptc1 knock-out mice in vivo and dysplasia in Barrett's esophagus and colonic adenomas during routine endoscopy. The specific aims of this research are as follows: 1) Measure the transverse and axial resolution, signal-to-noise, and field of view of the tabletop dual-angle-axis confocal prototype. 2) Collect reflectance and fluorescence images from cultured cancer cells expressing GFP/YFP. 3) Confirm the performance of the tabletop prototype with reflectance and fluorescence images collected from biopsy specimens of Barrett's epithelium and colonic adenomas, as a model of dysplasia. 4) Monitor the temporal and spatial extent of proliferation from medulloblastoma in post-natal ptc1 mice using the tabletop microscope. 5) Design and fabricate the miniaturized microscope using MEMS technology. 6) Monitor the temporal and spatial extent of proliferation from medulloblastoma in post-natal ptc1 knock-out mice and transplanted tumor cells using the miniaturized microscope. 7) Collect images from Barrett's epithelium and colonic adenomas during routine endoscopy.

描述（由申请人提供）：我们希望开发一种使用微型共焦显微镜进行体内成像的新方法，以在分子和细胞水平上研究肿瘤发展的生物学过程。 这种方法有可能彻底改变癌症研究。 该项目的长期目标是开发一种观察肿瘤如何起源、进展和侵袭的技术。 将通过用绿色或黄色荧光蛋白（GFP、YFP）标记的亚细胞成分的图像来监测细胞的行为。 微型共焦原型已经开发出来，其性能已通过离体图像得到证明。 我们计划通过采用双角轴架构的新设计来改进这款显微镜。 这种设计提供了改进的分辨率和工作距离，这将有利于体内成像。 然后，我们计划研究两种肿瘤模型，小鼠模型中的髓母细胞瘤与 ptc1 基因表达下降有关，以及巴雷特食管和结肠腺瘤中的发育不良。 我们将使用这些模型来开发新的显微镜。 首先，桌面双角轴原型的性能将通过表达 GFP/YFP 的培养细胞和组织切片的反射率和荧光图像以及巴雷特食管和结肠腺瘤的活检标本来表征。 然后，将从表达 GFP/YFP 的产后 ptc1 敲除小鼠中收集图像，以监测血管生成、肿瘤侵袭和对治疗的反应。 接下来，这些研究的结果将用于开发采用微机电系统（MEMS）制造技术重新设计的微型显微镜。 该微型显微镜将用于在常规内窥镜检查中研究ptc1基因敲除小鼠体内的髓母细胞瘤以及巴雷特食管和结肠腺瘤的发育不良。 本研究的具体目标如下： 1）测量桌面双角轴共焦原型机的横向和轴向分辨率、信噪比和视场。 2) 从表达 GFP/YFP 的培养癌细胞中收集反射率和荧光图像。 3) 使用从巴雷特上皮和结肠腺瘤的活检标本中收集的反射率和荧光图像来确认桌面原型的性能，作为不典型增生的模型。 4) 使用台式显微镜监测出生后 ptc1 小鼠髓母细胞瘤增殖的时间和空间范围。 5）利用MEMS技术设计和制造微型显微镜。 6) 使用微型显微镜监测出生后 ptc1 敲除小鼠和移植肿瘤细胞的髓母细胞瘤增殖的时间和空间范围。 7) 在常规内窥镜检查过程中收集 Barrett 上皮和结肠腺瘤的图像。