High accuracy quantum dot tracking in live cells

描述(申请人提供)：细胞(单细胞)层在身体的不同间隔之间形成屏障。分子在这些细胞层之间的运输受到高度调控。例如，脑和脑脊液(CSF)通过血脑屏障和血脑脊液屏障与血液分开。这些屏障促进了选定分子在不同隔间之间的运输，而它们阻止或严重限制了其他分子的运输。其他细胞层，如肠道上皮细胞，也有类似的功能。了解这些屏障上的细胞转运机制不仅对于理解这些基本的生物过程很重要，而且对于设计能够穿过这些屏障的治疗剂也是至关重要的。此外，阐明决定候选药物是否以及如何通过肿瘤细胞层进行运输的细胞过程是设计有效的抗癌疗法的基础。显微镜为研究亚细胞运输过程提供了一种自然的工具。然而，经典显微镜被设计为一次只能成像一个焦平面，即成像过程在很大程度上局限于与显微镜的焦平面相对应的两个维度。细胞屏障的过程本质上是三维的，因此通常不能在一个焦平面上成像。此外，现有的改变焦平面的方法通常太慢，不能遵循这些传输现象的高度动态和复杂的路径。这是目前关于蜂窝层内和跨蜂窝层的传输过程的有限知识的原因。为了克服与三维动态传输过程成像相关的问题，我们引入了多焦平面显微镜(MUM)。到目前为止，这项技术已经被广泛验证，可以对深度约为2.5微米的动力学进行成像。不幸的是，这个深度不足以描绘出这里感兴趣的细胞过程。因此，本申请致力于当前UM技术的扩展的开发，以使得能够成像在更深的蜂窝(单)层上成像传输过程所需的大得多的深度。这包括对这种最大单位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UM构型的实验测试中使用。量子点的高光稳定性使它们非常适合用于此类分析，这通常涉及到延长时间段的成像。具体目标是：具体目标1：开发用于厚细胞样品成像的多焦点显微镜方法。具体目标2：开发用于分析厚样本中的最大似然比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转运途径。

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.