High accuracy quantum dot tracking in live cells

描述（由申请人提供）：细胞（单层）层在身体的不同隔室之间形成屏障。分子穿过这些细胞层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。这些屏障促进了所选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。在这些障碍的细胞转运机制的知识不仅是重要的理解这些基本的生物过程，但也是至关重要的治疗药物，可以通过这些障碍的设计。此外，阐明决定候选药物是否以及如何穿过肿瘤细胞层的细胞过程是设计有效抗癌治疗剂的基础。 显微镜为亚细胞运输过程的研究提供了一种天然的工具。然而，经典的显微镜被设计成一次仅成像一个焦平面，即成像过程主要局限于与显微镜焦平面对应的两个维度。细胞屏障处的过程本质上固有地是三维的，因此通常不能在一个焦平面中成像。此外，改变焦平面的现有方法通常太慢，无法跟踪这些传输现象的高度动态和复杂的路径。这说明了目前关于细胞层中和跨细胞层的运输过程的知识有限。 为了克服与三维动态贩运过程的成像相关的问题，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，可用于高达约2.5微米深度的动态成像。不幸的是，这个深度不足以对这里感兴趣的细胞过程进行成像。因此，本申请致力于当前MUM技术的扩展的开发，以使得能够对跨更深的细胞（单）层的成像传输过程所需的显著更大的深度进行成像。这包括对这种MUM数据实施新的数据分析方法。量子点（QD）标记的蛋白质，如免疫球蛋白G分子，将在整个MUM配置的实验测试中使用。QD的高光稳定性使其非常适合用于此类分析，其通常涉及长时间成像。 具体目标1：开发用于厚细胞样本成像的多焦点显微镜方法。具体目标2：开发用于分析厚样品MUM数据的数据分析算法。具体目标3：阐明深层细胞样本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.