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: 6613204
• 负责人: Thomas J. Wang
• 金额: $ 11.72万
• 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/6613204
• 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(申请人提供)：我们希望开发一种新的体内成像方法，使用微型共聚焦显微镜在分子和细胞水平上研究肿瘤发展的生物学过程。这种方法有可能给癌症研究带来革命性的变化。该项目的长期目标是开发一种技术来观察肿瘤如何起源、发展和侵袭。细胞的行为将从用绿色或黄色荧光蛋白(GFP，YFP)标记的亚细胞成分的图像中进行监测。微型共聚焦样机已经研制成功，其性能已经用体外图像进行了验证。我们计划用一种使用双角度轴结构的新设计来改进这台显微镜。这种设计提供了更高的分辨率和工作距离，这将有助于活体成像。然后，我们计划研究两种肿瘤模型，一种是与ptc1基因表达降低相关的小鼠髓母细胞瘤模型，另一种是Barrett‘s食管腺瘤和结肠腺瘤中的异型增生。我们将使用这些模型来开发新的显微镜。首先，桌面双角度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和荧光图像以及Barrett‘s食管腺瘤和结肠腺瘤的活组织标本来表征。然后，将从出生后表达GFP/YFP的ptc1基因敲除小鼠身上收集图像，以监测血管生成、肿瘤侵袭和治疗反应。接下来，这些研究的结果将被用来开发采用微电子机械系统(MEMS)制造技术的重新设计的微型显微镜。这种微型显微镜将用于在活体内研究ptc1基因敲除小鼠的髓母细胞瘤，以及在常规内窥镜检查中巴雷特食道和结肠腺瘤的不典型增生。本研究的具体目的如下：1)测量桌面双角度轴共焦样机的横向分辨率、轴向分辨率、信噪比和视场。2)采集培养的表达GFP/YFP的癌细胞的反射和荧光图像。3)通过从Barrett上皮和结肠腺瘤活检标本收集的反射和荧光图像证实桌面原型的性能，作为异型增生的模型。4)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技术设计并制作了微型显微镜。6)用微型显微镜观察ptc1基因敲除小鼠和移植瘤小鼠髓母细胞瘤增殖的时间和空间范围。7)常规内窥镜检查时收集Barrett上皮和结肠腺瘤的图像。