Acquisition of a Multi-User Scanning Laser Confocal for Science and Engineering Research at Hopkins University

建议购买具有双光子能力的扫描激光共聚焦显微镜，以测量活细胞、组织、大分子组件和新材料的3D构象。使用这种新型显微镜，人们可以(I)从厚厚的荧光样品和光散射材料中切出干净的薄光学切片，(Ii)平行于视线观察样品平面，(Iii)深入到光散射组织、活细胞、复杂流体和复杂材料，以及(Iv)以非常高的分辨率获得三维视图。这种新的显微镜将补充霍普金斯大学教员目前使用的实验技术，他们活跃在生物医学工程、化学工程、材料科学和生物学的研究项目中。该提案详细说明了新的SLCM将产生重大影响的四个项目。这些项目包括：(1)开发与新的共聚焦显微镜相耦合的高分辨率粒子跟踪仪器，以探测生物聚合物的体外和体内机械性能；(2)研究上皮细胞膜蛋白的横向组织，重点是包装糖基磷脂酰肌醇锚定蛋白，以定向递送到形态极化细胞的顶端表面；(3)研究染色质绝缘体影响增强子-启动子相互作用的分子机制；特别是研究Gypsy绝缘体，位于果蝇反转录转座子5‘端转录非翻译区的序列；(4)以高空间和时间分辨率监测细胞和微细胞器转位过程。获得共焦显微镜的主要教育目标之一是培训霍普金斯大学的科学家和工程师使用生命和材料科学中最通用和最有前途的研究工具之一。当然，如果没有适当的训练，这样一种强大的工具就无法正确使用并充分发挥其潜力。因此，其中两名合作-PIS将教授一门名为“实用共焦显微镜”的新课程，该课程将在1月份的间歇期向有兴趣学习如何在研究生研究中使用共焦显微镜的理工科高级研究生提供。

It is proposed to purchase a scanning laser confocal microscope with two-photon capabilities to measure the 3D conformation of living cells, tissues, macromolecular assemblies, and novel materials. With this new microscope, one can (i) slice clean, thin optical sections out of thick fluorescent specimens and light scattering materials, (ii) view specimen planes parallel to the line of sight, (iii) penetrate deep into light-scattering tissues, living cells, complex fluids, and complex materials, and (iv) gain 3-D views at very high resolution. This new microscope will compliment the experimental techniques currently used by Hopkins faculty, active in research projects in biomedical engineering, chemical engineering, materials science and biology. This proposal details four projects where the new SLCM will have a dramatic impact. These projects include: (1) the development of a high resolution particle-tracking instrument coupled to the new confocal microscope to probe the in vitro and in vivo mechanical properties of biopolymers; (2) studying the lateral organization of membrane proteins in epithelial cells emphasizing the packaging of glycosylphos-phatidylinositol-anchored proteins, GPI-proteins, for targeted delivery to the apical surface of morphologically polarized cells; (3) studying the molecular mechanisms by which chromatin insulators affect enhancer-promoter interactions; in particular, studying the gypsy insulator, a sequence located in the 5' transcribed untranslated region of the gypsy retrotransposon of Drosophila; (4) monitoring the process of transfecting cells and micro-organelles translocations with high spatial and time resolution. One of the primary educational objectives for acquiring a confocal microscope is to train Hopkins scientists and engineers to use one of the most versatile and promising tools of investigation in the life and materials sciences. Of course, such a powerful instrument cannot be used properly and to its full potential with out proper training. Therefore, two of the Co- PIS will be teaching a new course entitled "Practical Confocal Microscopy" to be offered during the January intersession to advanced graduate students in science and engineering who are interested in learning how to use the confocal microscope in their graduate research.