High accuracy quantum dot tracking in live cells

描述（由申请人提供）：细胞内运输途径的阐明是细胞生物学的主要挑战，并有望导致对基本细胞过程的重要新见解。单分子方法的进步使人们期望蛋白质/大分子的运输机制不仅可以在大量群体中解开，甚至可以在单个分子水平上解开。由于量子点的光稳定性，可以对量子点标记的分子进行长时间的观察，这对于跟踪单个分子的细胞内通路是必要的。然而，目前的显微镜模式不太适合解决与三维细胞内运输有关的问题。传统的显微镜一次成像一个焦平面，而细胞是三维的物体，并且传输路径通常不局限于一个焦平面。再加上动态往往非常快，这意味着由于无法捕捉到贩运途径，因此往往不可能进行详细的贩运研究。另一个重要的问题是经典显微镜的低深度分辨能力。这意味着从图像中很难确定物体的三维位置，例如量子点标记的单个分子。这使得详细研究从内吞作用到早期核内体的途径或从分选核内体到胞吐作用的途径非常困难。为了解决这些问题，我们最近开发了一种新的成像方式，可以同时对不同的焦平面成像。这种模式包括用全内反射荧光显微镜在质膜上成像事件的能力，同时在更高焦平面上使用表观荧光模式捕捉细胞内部的过程。使用这种方法，量子点标记的蛋白质可以成像，因为它们遵循通过细胞的三维途径。同样重要的是，这种方法可以克服深度歧视问题。这个项目的一个中心方面将是开发算法，用它可以识别单个分子的三维位置。提出的方法将在一个重要的运输问题上进行测试，即量子点标记的免疫球蛋白G分子的细胞内运输、内吞和胞吐。我们的具体目标是：1。开发分析工具以确定细胞中量子点标记蛋白的三维位置。2. 分析QD标记蛋白的三维位置确定的准确性。3. 在实验数据上验证所提出的算法。公共卫生相关性：我们建议开发一种活细胞成像的新技术。这项技术有望克服现有方法的重大限制，这些限制迄今为止阻碍了研究人员研究细胞功能的核心方面。这项新技术将允许进行重要的研究，以增加我们对细胞功能的理解。值得注意的是，这项技术还将改进研究人员可用的工具，以研究快速扩展的一类治疗方法，即基于抗体的药物如何与细胞相互作用。

DESCRIPTION (provided by applicant): The elucidation of intracellular trafficking pathways is a major challenge in cell biology and promises to lead to significant new insights into basic cellular processes. The advances in single molecule methodologies hold the expectation that the trafficking mechanisms for proteins/macromolecules can be unraveled not only for bulk populations, but even at the level of individual molecules. Due to the photo-stability of quantum dots (QDs) it is possible to observe QD-labeled molecules for extended observation periods, which is necessary to follow intracellular pathways of individual molecules. However, current microscopy modalities are not well suited to address problems related to intracellular trafficking in three dimensions. Whereas classical microscopes image one focal plane at a time, cells are three dimensional objects and the trafficking pathways are not typically restricted to one focal plane. This, combined with the fact that the dynamics are often very fast, means that detailed trafficking studies are often not possible since the pathways cannot be captured. A further significant problem is the low depth discrimination capability of a classical microscope. This means that from an image it is very difficult to determine the three dimensional position of an object such as a QD-labeled single molecule. This makes it highly problematic to study in detail processes such as the pathway from endocytosis to early endosome or the pathway from sorting endosome to exocytosis. To address these problems we have recently developed a new imaging modality with which different focal planes can be imaged at the same time. This modality includes the capability to image events at the plasma membrane with total internal reflection fluorescence microscopy, whilst simultaneously capturing processes in the interior of the cell using epifluorescence mode in higher focal planes. Using this approach QD-labeled proteins can be imaged as they follow a three dimensional pathway through a cell. Equally important is the promise that the depth discrimination problem can be overcome with this approach. A central aspect of this project will be to develop algorithms with which the three dimensional location of a single molecule can be identified. The proposed approaches will be tested on an important trafficking problem, i.e. the intracellular trafficking, endocytosis and exocytosis of QD-labeled immunoglobulin G molecules. Our Specific aims are: 1. To develop analytical tools to determine the 3D location of a QD-labeled protein in a cell. 2. To analyze how accurately the 3D position of a QD labeled protein can be determined. 3. To test the proposed algorithms on experimental data. PUBLIC HEALTH RELEVANCE: We propose to develop a new technology for the imaging of living cells. This technology promises to overcome significant limitations in existing approaches which have to date prevented researchers from studying central aspects of the functioning of cells. The new technology will permit investigations that are important to increase our understanding of how cells function. Significantly, this technology will also improve the tools that researchers have available to investigate how a rapidly expanding class of therapeutics, namely antibody-based drugs, interact with cells.