Bright Blinking Probes for Fast Multicolor Superresolution Imaging in Live Cells

我们的目标是开发新的探测器和方法，允许直接测量空间- 活细胞中蛋白质的时间行为。使用用来装饰的多功能树状大分子 两种染料，一种荧光供体阵列和一种荧光猝灭染料，我们将开发非常高的 由小的融合蛋白标签激活的亮度探针。因为这些元素的绑定 模块产生间歇性信号，我们将把这些探头集成到一个新的 超分辨率成像方法，使分子尺度定位和跟踪所有 通过在平衡点的随机激活来标记样本区域中的蛋白质。我们将对这些进行验证 肾上腺素能受体和细胞骨架动力学研究中的新探针和新方法。 总的来说，该计划将开发、验证和交付新的成像工具和方法，以 在纳米尺度上跟踪标记蛋白质的位置和动态。总而言之，这些 方法预计将在单个 动态单分子的分子亮度、定位精度和时间尺度 在活细胞中进行测量。

We aim to develop new probes and methods that will allow direct measurements of the spatio- temporal behavior of proteins in living cells. Using multifunctional dendrimers decorated with two dyes, a fluorescent donor array and a fluorogenic quencher dye, we will develop very high brightness probes that are activated by small fusion protein tags. Because the binding of these modules produces an intermittent signal, we will integrate these probes into a new superresolution imaging method that enables molecular scale localization and tracking of all tagged proteins in a sample region by stochastic activation at equilibrium. We will validate these new probes and methods in studies of ¿2 adrenergic receptor and cytoskeletal dynamics. Collectively, this program will develop, validate, and deliver new imaging tools and methods to track positions and dynamics of tagged proteins at nanometer length scales. Collectively, these methods are anticipated to deliver at least an order of magnitude improvement in the single molecule brightness, the localization accuracy, and the timescale of dynamic single molecule measurements in living cells.