Multiplexing Quantitative Photostable Nanoscopy for Single Live Cell Imaging

项目摘要（摘要）

Project Summary (Abstract) Multiplexing Quantitative Photostable Nanoscopy for Single Live Cell Imaging Single cells are building-block of all living organisms. Different types of cells express trace amounts of distinctive sets of receptors, which can serve as biomarkers for cell identification and for disease diagnosis. Notably, cell populations often exhibit high heterogeneity. Rare subsets of single live cells can act distinctively and they can affect and alter functions of an entire cell population over time. Thus, it is vital to develop new tools that can map the heterogeneity of a cell population at single cell resolution in situ and with spatiotemporal resolutions over time, in order to understand the functions of rare subsets of single cells and their communications with neighboring cells, and how they evolve from healthy states to pathological states over time. Current methods (flow cytometry, genetic analysis methods and fluorescence-based imaging and assays including high-content screening, HCS) that are used to identify specific single live cells cannot offer sufficient photostability, multiplexing capability, selectivity, single molecule sensitivity, temporal and spatial resolutions to specifically study individual receptor molecules on single live cells at its native environments and characterize their functions in situ and over a long period of time (days). Rare subsets of single cells often need to be isolated or pre-concentrated for further analysis, which loses the opportunity to study them continuously in its native environments over time. We propose to develop multicolored multifunctional photostable single-molecule nanoparticle optical biosensors (m2-SMNOBS) and far-field multiplexing quantitative photostable optical nanoscopy (mq-PHOTON) for quantitatively imaging of multiple receptors on single live cells, aiming to identify single live cells, especially rare subsets of single live cells (≤ 5%) in highly heterogeneous cell populations and study their functions in the cell population in situ over time with spatiotemporal resolutions. To demonstrate unique capabilities of these new tools, as a proof-of-concept, we will use these new tools to detect single brain cancer stem cells (bCSCs) in highly heterogeneous brain tumor cells and study their differentiation in their native environment over time (48 hours) with spatiotemporal resolutions. The outcomes of the proposed research include the development of highly innovative tools for molecular identification and characterization of functions of rare and vital subsets of single live cells in highly heterogeneous cell populations with spatiotemporal resolutions in situ in real time at single molecule resolution. These powerful new tools will become extremely valuable to address a wide range of pressing biochemical and biomedical questions about molecular and real-time characterization of functions of single receptors and biomarkers on single live cells in situ over time.