High-throughput and highly multiplexed imaging with programmable nucleic acid probes

A method for spatial imaging a plurality of protein targets in situ within a sample comprising cells and/or tissue comprising:

• (1) contacting the sample comprising a plurality of targets with a plurality of target-specific binding partners, wherein each of the target-specific binding partners is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,
• (2) contacting the sample with labeled imager strands, wherein the labeled imager strands comprise a sequence that hybridizes to the docking strand a moiety cleavable by a nucleic acid-DNA glycosylase and a fluorescent label,
• (3) imaging the sample to detect a fluorescent signal from the hybridized imager strands,
• (4) adding a nucleic acid-DNA glycosylase to the sample and,
• removing the fluorescent signal from the hybridized imager strands, and
• (5) repeating steps (2) to (4), each time with a labeled imager strand having a unique nucleotide sequence relative to all other labeled imager strands, whereby a plurality of targets are imaged in situ.