C 2014 American Chemical Society Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

ife is orchestrated by programmable biomolecules interacting within com-plex biological circuits. One of the central technical challenges in biological research is the difficulty in interrogating the state of these circuits within intact organisms. In this pursuit, in situ hybridiza-tion methods provide biologists with a powerful tool for mapping mRNA expres-sion in a morphological context. However, with traditional approaches, it remains chal-lenging to simultaneously map the expres-sion patterns of multiple target mRNAs within a single intact vertebrate embryo, hindering the study of development and disease in model systems most relevant to human biology. With in situ hybridization, nucleic acid probes are used to detect complementary nucleic acid targets within fixed samples; subsequent washes remove unbound probes prior to imaging of fluorophores or chromophores that either label the probes directly or are localized in the vicinity of probes during a subsequent amplification step.1,2 Direct-labeled fluorescent probes are well suited for multiplexing,39 but do not generate sufficient signal-to-background for general-purpose use within intact verte-brate embryos. To improve the signal-to-background ratio, significant effort has been devoted to the development of in situ amplification methods.6,1021 In the demanding imaging environment ofwhole-mount vertebrate embryos, traditional ap-proaches employ enzymes to catalyze the deposition of reporter molecules in the vicinity of nucleic acid probes.2225 These methods are widely used despite significant drawbacks. Spatial resolution is typically compromised by diffusion of reporter mol-ecules prior to deposition,26,27 and the lack of orthogonal deposition chemistries dic-tates that in situ amplification be performed serially for multiplexed studies,24,25,28,29 leading to progressive sample degradation * Address correspondence t