Single-molecule imaging with super-resolution

描述（由申请人提供）：拟议工作的目标是推动生物成像的边界，以便能够常规地以特殊的空间分辨率（2-3纳米）和高灵敏度（即可以检测到单个分子）对特定细胞分子进行成像。如果我们成功了，我们的技术将允许任何细胞生物学家以前所未有的精确度研究活细胞内的任何生物过程。目前的技术允许在体外检测单分子，但不能常规地在活细胞内检测，亚衍射远场光学成像目前可以在固定细胞上以20-30纳米的分辨率进行，但不能在活细胞上进行。在我们看来，改善单分子和超分辨率成像的主要挑战是缺乏合适的荧光探针。我们计划分三个部分来解决这个问题。首先，我们将设计和合成一种新的荧光探针，这种荧光探针具有极高的亮度和光稳定性，而且还可以传递到活细胞的细胞质中并保持良好的溶解性。其次，我们将开发将这些荧光团与感兴趣的特定细胞蛋白结合的新方法。第三，我们将开发物理方法来增强现有荧光团的荧光发射，同时最大限度地减少细胞自身荧光的背景。所有这些新分子和技术将共同应用于对固定细胞和活细胞进行超灵敏、高分辨率的光学成像。我们对神经科学也有特别的兴趣，并将应用我们开发的新的光学成像方法来研究在学习和记忆形成中起核心作用的神经递质受体的运输和定位。公共卫生相关性：如果成功，我们的研究将产生成像技术，通过允许以前所未有的细节对活细胞中的生物过程进行单分子和超分辨率光学成像，将使整个细胞生物学家社区受益。如果我们提出的神经递质受体成像实验成功，我们将对学习、记忆和神经元发育的分子机制有基本的了解，这将反过来揭示神经和精神疾病，包括中风、癫痫、脑损伤、成瘾、精神分裂症、自闭症和慢性疼痛。

DESCRIPTION (provided by applicant): 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. We also have a special interest in neuroscience, and will apply the new optical imaging methods that we develop to study the trafficking and localization of neurotransmitter receptors that play a central role in learning and memory formation. Public Health Relevance: If successful, our research will produce imaging technologies that will benefit the entire community of cell biologists, by allowing single-molecule and super-resolution optical imaging of biological processes in living cells with unprecedented detail. If our proposed experiments on neurotransmitter receptor imaging are successful, we will gain fundamental insight into the molecular mechanisms of learning and memory and neuronal development, which will in turn shed light on neurologic and psychiatric disorders including stroke, epilepsy, brain injury, addiction, schizophrenia, autism, and chronic pain.