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

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates thathave an appropriate size for long circulation and diffusion and show virtually no nonspecificbinding to cells/serum while binding to cells of interest with high specificity. In addition, theseconjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, weshow that this can be achieved using compact (∼15 nm in hydrodynamic diameter) andbiocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbsto QDs coated with norbornene-displaying polyimidazole ligands using tetrazine–norbornenecycloaddition. 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-Abconjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rarepopulations 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 functionalimaging to study the interactions between cells and between a cell and its environment in intact anddiseased tissues.Studying migration of individual endogenous cells and their interactionswith the surrounding microenvironment in vivo would greatly help expand our knowledge on how cellsbehave in their complex native biological network. However, single-cell imaging of a rare populationin vivo places highly stringent constraints on targeting fluorophores. First, identifying specificcell populations among various types of cells expressing overlapping surface markers demandssimultaneous labeling of multiple markers, and therefore requires fluorophores with narrow emissionfeatures. Second, the size of targeting fluorophore conjugates needs to be optimized tosimultaneously achieve both long blood circulation times and high diffusion in dense in vivoenvironments. Third, because only a small fraction of systemically administered fluorophores isdelivered to the targeting sites, resulting in low signal, targeting fluorophore conjugates mustemit bright signals and exhibit minimal nonspecific binding to serum proteins and cells. Fourth,targeting fluorophore conjugate samples must be free of unbound targeting molecules, because unboundtargeting molecules can block target sites and yield decreased signal.Because of the lack of such a technology, direct labeling of single cells from an endogenous rarecell population in live and nonmanipulated animal models has not been possible. Instead, researchershave used (i) immunohistochemistry (1),(ii) ex vivo (2) or intravital imaging (3) of injected target cells, which are fluorescently labeled exvivo, or (iii) ex vivo (4) or intravitalimaging (5, 6) of targetcells that are genetically modified to express fluorescent markers. However, none of these methodsreproduce the native microenvironment of target cells. Tissue immunohistochemistry and ex vivoimaging only provide a snapshot image of a perturbed state. Intravital imaging allows for real-timeimaging but requires either genetically engineered mouse models to induce endogenous expression offluorescence proteins in cells or irradiation of mice for marrow depletion and repopulation withsystemically infused cells.Quantum dots (QDs) possess unique optical properties that are ideal for in vivo imaging in liveanimals. Specifically, QDs have a tunable band gap ranging from the visible to the IR, high quantumyields (QYs), narrow and symmetric emission features, broad absorption above the band gap, largemultiphoton absorption cross-sections, and high photostability (7–10). These properties make QDs amenable to optical multiplexing forsimultaneous study of various targets, long-term tracking, and deep-tissue imaging using multiphotonmicroscopy. Despite these exciting capabilities, most imaging studies using QDs have involved eitherin vitro targeting or ensemble measurements of QD signals over large volumes of tissue in vivo(11–15). Thisrestriction is caused by a lack of technology for synthesizing QD conjugates that are optimal forsingle-cell imaging in vivo.Here, we report the development of QD-Ab conjugates that satisfy all of the constraints describedabove. Moreover, we used these QD-Ab conjugates for in vivo cytometry of endogenous bone marrowcells (BMCs) in their unperturbed microenvironment. Our technique provides opportunities to studythe movements of single cells and the interactions between cells and between a cell and itsenvironment in their native states.