Single-molecule imaging with super-resolution

拟议工作的目标是推动生物成像的边界， 这将是常规可能成像特定的细胞分子与特殊的 空间分辨率（2 - 3nm）和高灵敏度（即，可以检测单个分子）。如果 如果我们成功了，我们的技术将允许任何细胞生物学家研究任何生物学， 在活细胞内，以前所未有的准确性进行。目前的技术允许 在体外检测单个分子，但在活细胞内不常规检测， 衍射远场光学成像目前可以在20 - 30 nm分辨率下进行， 固定的，但不是活细胞。在我们看来，改善单分子生物学的主要挑战 和超分辨率成像是缺乏合适的荧光探针。我们计划解决 这个问题分为三个部分。首先，我们将设计和合成新的荧光探针 它们非常明亮，对光稳定，但也可以被输送到细胞质中， 并保持良好的溶解性。其次，我们将开发新的方法， 将这些荧光团与感兴趣的特定细胞蛋白质结合。三是 开发物理方法来增强现有的荧光发射， 荧光团，同时最小化来自细胞自发荧光的背景。所有这些新 分子和技术将共同应用于执行超灵敏，高 固定和活细胞上的分辨率光学成像。

The objective of the proposed work is to push the boundaries of biological imaging, so that it will be routinely possible to image specific cellular molecules with exceptional spatial resolution (2-3 nm) and high sensitivity (i.e., single molecules can be detected). If we are successful, our technologies will allow any cell biologist to study any biological process, inside living cells, with unprecedented accuracy. Current technology allows detection of single molecules in vitro but not routinely inside living cells, and sub- diffraction far-field optical imaging can currently be performed at 20-30 nm resolution on fixed, but not living cells. In our view, the major challenge to improving single-molecule and super-resolution imaging is the lack of suitable fluorescent probes. We plan to solve this problem in three parts. First, we will design and synthesize new fluorescent probes which are extremely bright and photostable, but can also be delivered into the cytoplasm of living cells and remain well-solubilized. Second, we will develop novel methods for conjugating these fluorophores to specific cellular proteins of interest. Third, we will develop physical methods for enhancing the fluorescence emission of existing fluorophores, while minimizing background from cellular autofluorescence. All these new molecules and techniques will be collectively applied to perform ultra-sensitive, high resolution optical imaging on fixed and living cells.