Quantum dot/antibody conjugates for in vivo cytometric imaging in mice

Significance One of the key questions in biology is understanding how cells move, interact, and evolve in living organisms. Tremendous efforts have been made to answer these questions in vitro, which have yielded a molecular-level understanding of cellular events. However, an increasing number of studies indicate that cellular activities need to be understood in the context of their natural environments. Single-cell labeling methods in use currently involve immunohistochemistry, genetic manipulation, or irradiation of mice, none of which reflect the native microenvironments. Here, we report quantum dot immunoconstructs that can be used for intravital imaging of single cells in unmanipulated mice and multiplexed in vivo cytometric analysis of rare cell populations. Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine–norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1+c-Kit+ cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.

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