Novel Confocal Microscope for Molecular/Cellular Imaging

描述（由申请人提供）：我们想开发一种新的方法，利用微型共聚焦显微镜在体内成像，从分子和细胞水平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发展一种技术来观察肿瘤的起源、发展和侵袭。细胞的行为将通过标记有绿色或黄色荧光蛋白（GFP， YFP）的亚细胞成分图像来监测。一个小型化的共聚焦原型已经开发出来，其性能已经用离体图像证明。我们计划用一种新的设计来改进这种显微镜，使用双角轴结构。这种设计提供了更高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：与ptc1基因表达降低相关的小鼠模型髓母细胞瘤，以及Barrett食管和结肠腺瘤的发育不良。我们将利用这些模型来研制新的显微镜。首先，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食管和结肠腺瘤的活检标本来表征桌面双角轴原型的性能。然后，收集出生后表达GFP/YFP的ptc1敲除小鼠的图像，以监测血管生成、肿瘤侵袭和对治疗的反应。接下来，这些研究结果将用于利用微机电系统（MEMS）制造技术开发重新设计的小型化显微镜。该微型显微镜将用于ptc1基因敲除小鼠体内成神经管细胞瘤的研究，以及常规内镜下Barrett食管和结肠腺瘤发育不良的研究。本研究的具体目的如下：1)测量台式双角轴共聚焦样机的横向和轴向分辨率、信噪比和视场。2)收集表达GFP/YFP的癌细胞的反射率和荧光图像。3)利用Barrett上皮和结肠腺瘤活检标本的反射和荧光图像证实桌面原型作为不典型增生模型的性能。4)利用桌面显微镜观察ptc1小鼠髓母细胞瘤增殖的时空范围。5)利用MEMS技术设计制作微型化显微镜。6)利用微型显微镜监测ptc1基因敲除小鼠和移植肿瘤细胞髓母细胞瘤的时空增殖程度。7)在常规内镜检查中采集Barrett上皮和结肠腺瘤的图像。

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.