Programmable In Situ Amplification for Multiplexed Bioimaging

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously detect multiple target mRNAs in vertebrate embryos and tissue sections – a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. This thesis presents a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. Robust performance and high signal-to-background are achieved when imaging five target mRNAs at the same time in fixed whole-mount zebrafish embryos. The programmability and sequence specificity of these amplification cascades enable all five amplifiers to operate orthogonally at the same time in the same sample. The fact that amplification polymers are triggered to self-assemble in situ results in excellent sample penetration and high signal-to-background. These properties suggest the broad applicability of fluorescent in situ HCR amplification to multiplexed imaging of mRNA expression in normal and pathological cells, embryos, and tissue sections.