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 traffics 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 test the methodology on thick cellular samples.
We propose to continue the development of a new microscopy 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 transport of molecules; such as therapeutics; in cells and the functioning of cells. Significantly; this further development 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 and especially how they are transported across layers of cells that form natural boundaries in the body.
|Velmurugan, Ramraj; Ramakrishnan, Sreevidhya; Kim, Mingin et al. (2018) Phagocytosis of antibody-opsonized tumor cells leads to the formation of a discrete vacuolar compartment in macrophages. Traffic 19:273-284|
|Ward, E Sally; Ober, Raimund J (2018) Targeting FcRn to Generate Antibody-Based Therapeutics. Trends Pharmacol Sci 39:892-904|
|Chao, Jerry; Velmurugan, Ramraj; You, Sungyong et al. (2017) Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context. Proc SPIE Int Soc Opt Eng 10070:|
|Vahid, Milad R; Chao, Jerry; Ward, E Sally et al. (2017) A state space based approach to localizing single molecules from multi-emitter images. Proc SPIE Int Soc Opt Eng 10070:|
|Chao, Jerry; Ram, Sripad; Ward, E Sally et al. (2016) Investigating the usage of point spread functions in point source and microsphere localization. Proc SPIE Int Soc Opt Eng 9713:|
|Chao, Jerry; Sally Ward, E; Ober, Raimund J (2016) Fisher information theory for parameter estimation in single molecule microscopy: tutorial. J Opt Soc Am A Opt Image Sci Vis 33:B36-57|
|Velmurugan, Ramraj; Challa, Dilip K; Ram, Sripad et al. (2016) Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells. Mol Cancer Ther 15:1879-89|
|Ober, Raimund J; Tahmasbi, Amir; Ram, Sripad et al. (2015) Quantitative Aspects of Single Molecule Microscopy. IEEE Signal Process Mag 32:58-69|
|Tahmasb, Amir; Ward, E Sally; Ober, Raimund J (2015) New results on the single molecule localization problem in two and three dimensions. Proc SPIE Int Soc Opt Eng 9554:|
|Chao, Jerry; Ram, Sripad; Lee, Taiyoon et al. (2015) Investigation of the numerics of point spread function integration in single molecule localization. Opt Express 23:16866-83|
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