Assistant Professor University of Pennsylvania, United States
Introduction:: The ability to observe cells in live organisms is essential for understanding their functions and interactions. Temporal processes are often studied by introducing fluorescent proteins coupled to a promoter, protein or biological compartment of interest, making otherwise invisible physiologic events observable by fluorescence microscopy. This strategy has been intensely successful in cell biology. However, a major challenge has been the limited ability to perform higher multiplexing beyond four to six colors due to an inherent spectral overlap in fluorescence microscopy. To solve this limitation, we developed a click chemistry-based strategy that enables ultra-fast multiplexed live cell imaging in vitro, ex vivo, and in vivo.
Materials and Methods:: NHS-TCO-Flurophores (AF488, AF594, AF647) were conjugated to antibodies and tetrazine (Tz) was conjugated to black hole quenchers (BHQ3). Fine needle aspirates, cancer cell lines (A431, A549), peripheral blood mononuclear cells, freshly harvested mouse liver tissues, patient-derived brain organoids, and dorsal skinfold window chamber mouse model were used in this study. Fluorescence microscopy, flow cytometry, and plate reader were used for molecular measurements.
Results, Conclusions, and Discussions:: Tz–TCO click reactions reached >99% quenching in seconds, allowing a cyclic imaging cycle to be completed within a few minutes. This bioorthogonal cycling was performed at non-toxic nanomolar concentrations while achieving the high signal to background needed for serial profiling. Using this technology, we performed 4-color imaging of living mouse peripheral blood mononuclear cells (PBMCs), multiplexed imaging experiments in living murine hepatic tissue and visualize myeloid subsets in multicolor staining of living murine bone marrow. We also demonstrated the ability of serial bioorthogonal cycling to track the differentiation of immortalized hematopoietic progenitor cells into neutrophils in longitudinal profiling of the same cells over 6 days. This technology was further applied to profile live patient-derived glioblastoma organoids with multiple markers ( >9) and to monitor immunotherapy response by adding CAR-T cells to the organoid culture. A dorsal skinfold window chamber mouse model was also used to show its feasibility of in vivo multiplexed live cell imaging with antibodies.