MRI: Development of FPALM-STORM for Live Cell Single Molecule Microscopy

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。蛋白质是在细胞内发现的纳米级(十亿分之一米)机器。为了看到如此微小的物体，我们需要显微镜。电子显微镜具有很好的分辨率，可以对这样的小物体进行可视化，但样本(通常是细胞或其他生物元素)必须是固定的或冷冻的，因此无法可视化活细胞。另一方面，光学显微镜，如荧光显微镜，允许蛋白质在活组织中定位。但是，由于光使纳米物体看起来像是直径为100纳米的模糊圆点，运动动力学方面的成果在分辨率上丢失了。此外，细胞内有数以千计的相同类型的蛋白质，所以模糊的小点让位于模糊的斑点，而模糊的小斑点更难分辨。该方案旨在利用荧光光活化定位显微镜(Palm)和随机光学重建显微镜(STORM)大大提高光学分辨率。在这些技术中，我们使用特殊的荧光分子，可以一次打开只显示极少数蛋白质。每个单独的荧光标记蛋白质可以使用数学拟合例程进行高精度的定位。这些运动也可以在细胞内追踪。这些分子被关闭，一组新的分子被打开、定位和跟踪。分析程序将建立所有分子的位置图，以揭示详细的结构数据。这一新仪器将揭开细胞史无前例的新视角，并揭示细胞工作的科学真相。马萨诸塞大学阿默斯特分校的研究科学家和学生将使用这种仪器更深入地研究一些基本的生物学问题。例如，几个小组对细胞分裂过程感兴趣，这是器官发育、组织和器官再生以及癌细胞生长的基本过程。一些小组专注于植物细胞的生长，这可能会对未来的生物能源产生影响。另一组研究神经感受器动力学以了解记忆的形成。这些对细胞的新一瞥将为基础科学研究带来新的信息，并揭示细胞内生命的惊人图像。研究人员将在马萨诸塞大学物理系托管的画廊网页(www.umass.edu/phy)上展示这些图像。此外，该大学将在展示细胞的大幅面照片时与阿默斯特社区分享这些图像。这些指纹将通过可视化程序在Amherst地区及其周围展示，包括机动车和图书馆部门。这些照片将包括对被成像的蛋白质的描述，以及图像所呈现的整体科学。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Proteins are nanometer-scale (one billionth of a meter) machines found inside of cells. In order to see such tiny objects we need microscopy. An electron microscope has good resolution to allow visualization of such small objects, but the samples (usually cells or other biological elements) must be fixed or frozen, so visualizing live cells is not possible. On the other hand, light microscopy, such as fluorescence microscopy, allows for proteins to be localized in living tissue. But, the gains in motion dynamics are lost in resolution, since light makes nanometer objects appear as fuzzy dots that are 100s of nanometers across. In addition, there are thousands of proteins of the same type inside cells, so fuzzy dots give way to fuzzy blobs that are even more difficult to resolve. This proposal aims to greatly enhance the optical resolution using Fluorescence Photoactivation Localization Microscopy (PaLM) and Stochastic Optical Reconstruction Microscopy (StORM). In these techniques, we use special fluorescent molecules that can be switched on to visualize a very few proteins at a time. Each individual fluorescently labeled protein can be localized with high accuracy using mathematical fitting routines. The motions can also be tracked inside cells. These molecules are switched off, and a new set of molecules are switched on, localized, and tracked. The analysis routines will build a map of the locations of all the molecules to reveal detailed structural data. This new instrument will unlock unprecedented new views of cells and scientific revelations of cellular workings. The research scientists and students at the University of Massachusetts Amherst will use this instrument to delve deeper into some basic biological questions. For instance, several groups are interested in the cell division process, which is the underlying process for organ development, tissue and organ regeneration, and cancer cell growth. A few groups focus on plant cell growth, which may have future implications for bio-energy. Another group studies neuron-receptor dynamics to understand memory formation. These new glimpses of cells will result in new information for the study of basic science and reveal amazing images of life inside a cell. Researchers will display these images obtained on a gallery webpage hosted by the University of Massachusetts Department of Physics (www.umass.edu/physics). In addition the University will share these images with the Amherst community in a display of large-format prints of the cells. The prints will be displayed in and around the Amherst area, including the Department of Motor Vehicles and Libraries through the VISUAL program. The prints will include descriptions of the proteins being imaged and the overall science being presented by the image.