High accuracy quantum dot tracking in live cells

描述（由申请人提供）：细胞（单层）层形成身体不同隔室之间的屏障。分子在这些细胞层之间的运输受到高度调节。例如，脑和脑脊液（CSF）通过血脑屏障和血-CSF屏障与血液分离。这些屏障促进了选定分子在不同隔室之间的运输，而它们阻止或严重限制了其他分子的运输。其他细胞层如肠上皮细胞也有类似的功能。了解这些屏障处的细胞转运机制不仅对理解这些基本的生物学过程很重要，而且对设计能够穿过这些屏障的治疗剂也至关重要。此外，阐明决定候选药物是否以及如何通过肿瘤细胞层的细胞过程是设计有效抗癌疗法的基础。显微镜为亚细胞运输过程的调查提供了一个自然的工具。然而，经典的显微镜被设计成一次只能成像一个焦平面，也就是说，成像过程主要局限于与显微镜焦平面相对应的两个维度。细胞屏障处的过程本质上是三维的，因此通常不能在一个焦平面内成像。此外，现有的改变焦平面的方法通常太慢，无法遵循这些传输现象的高度动态和复杂的途径。这解释了目前关于细胞层内和细胞层间运输过程的有限知识。为了克服三维动态贩运过程成像的问题，我们引入了多焦平面显微镜（MUM）。迄今为止，该技术已被广泛验证，可用于深度约2.5微米的动态成像。不幸的是，这个深度不足以成像这里感兴趣的细胞过程。因此，当前的应用致力于开发当前MUM技术的扩展，以实现更大深度的成像，这是跨更深细胞（单细胞）层成像传输过程所需的。这包括对此类MUM数据实施新颖的数据分析方法。量子点（QD）标记的蛋白质，如免疫球蛋白G分子，将在整个MUM结构的实验测试中使用。量子点的高光稳定性使它们非常适合用于这种通常涉及长时间成像的分析。具体目标是：具体目标1：发展多焦点显微镜方法成像厚细胞样品。具体目标2：开发用于厚样本MUM数据分析的数据分析算法。特异性目的3：阐明深层细胞样品中的单分子运输途径。

DESCRIPTION (provided by applicant): Cellular (mono)layers form barriers between different compartments of the body. The transport of molecules across these cell layers is highly regulated. For example, the brain and cerebrospinal fluid (CSF) are separated from the blood by the blood-brain barrier and the blood-CSF barrier. These barriers facilitate the transport of selected molecules between the different compartments, whereas they prevent or severely limit the transport of others. Other cell layers such as gut epithelial cells serve similar functions. Knowledge of the cellular transport mechanisms at these barriers is not only important for understanding these fundamental biological processes but is also critical for the design of therapeutic agents that can pass through these barriers. Further, elucidation of the cellular processes that determine whether and how a drug candidate trafficks through the cell layers of a tumor is fundamental to the design of effective anti-cancer therapeutics. Microscopy provides a natural tool for the investigation of subcellular trafficking processes. However, classical microscopes are designed to image only one focal plane at a time¿ i.e. to image processes that are largely confined to the two dimensions corresponding to the focal plane of the microscope. The processes at cellular barriers are inherently three dimensional in nature and therefore typically cannot be imaged in one focal plane. In addition, the available methods for changing focal planes are usually too slow to follow the highly dynamic and complex pathways of these transport phenomena. This accounts for the limited knowledge that is currently available regarding the transport processes in and across cellular layers. To overcome the problems associated with the imaging of dynamic trafficking processes in three dimensions, we have introduced multifocal plane microscopy (MUM). To date this technology has been extensively validated for imaging of dynamics up to a depth of around 2.5 micrometers. Unfortunately, this depth is insufficient to image the cellular processes that are of interest here. Therefore the current application is devoted to the development of extensions of the current MUM technology to enable the imaging of significantly larger depths that are required to image transport processes across deeper cellular (mono)layers. This includes the implementation of novel data analysis approaches for such MUM data. Quantum dot (QD)-labeled proteins, such as immunoglobulin G molecules, will be used throughout the experimental testing of the MUM configurations. The high photostability of QDs makes them very well suited for use in such analyses, which typically involve imaging over extended time periods. The Specific Aims are: Specific Aim 1: To develop multifocal microscopy approaches for the imaging of thick cellular samples. Specific Aim 2: To develop data analysis algorithms for analysis of MUM data from thick samples. Specific Aim 3: To elucidate single molecule trafficking pathways in deep cellular samples.