High multiplexing capability is indispensable for high-content mapping. However, spectral overlap and lack of
orthogonal labeling create severe limitations for conventional fluorescence imaging. For protein imaging, these
limitations have been circumvented through multiplexed detection via unconventional probes and specialized
instruments, iterative sequential antibody labeling and imaging, or sequential detection through DNA-barcoding.
Although all of these techniques can theoretically achieve high multiplexing, they come at a cost of limited
throughput. Utilizing in situ signal amplification would substantially reduce exposure - and thus imaging - time per
frame, allowing for high throughput as well as improved sensitivity. Although amplification methods exist, they have
not been robustly multiplexed beyond 5-8 spatially overlapping targets. A critical unmet technical need for fully
realizing the HuBMAP vision is a highly multiplexed signal amplification technique that can simultaneously amplify
tens of distinct targets by tens- to hundreds-fold, thereby enabling highly multiplexed, high throughput, in situ
imaging of proteins in human tissues. We propose such an in situ signal amplification method, based on a novel
molecular mechanism that we recently published. In the new method, staining with multiple probes (DNA-barcoded
primary antibodies) will be performed simultaneously, and then all barcodes will be simultaneously extended into long concatemers in situ. Finally, mapping of concatemers will be sequentially performed through rapid exchange and imaging cycles, improving throughput by 10-fold while enabling detection of rare targets in tissues. Beyond proteins, the method will be applicable to RNA and DNA (chromosome) targets. We will integrate the method with
commercially available, automated staining and imaging systems, and apply it to image diverse human tissues via
broad collaboration within and beyond HuBMAP community.
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