Applications and development of in situ nucleic acid visualization techniques

In situ nucleic acid visualization techniques are important tools and methods for studying the functions of nucleic acid molecules (DNA and RNA) and are widely used in various fields, including the spatial localization of gene expression, the influence of nucleic acid molecules on biological processes, and pathological diagnosis. On the basis of their mechanisms, these techniques can be categorized into direct detection methods, such as fluorescence in situ hybridization signal amplification-based techniques, such as Hybridization Chain Reaction (HCR), rolling circle amplification (RCA) and RNAscope; and combined coding and high-throughput imaging approaches, such as sequential Fluorescence In Situ Hybridization (seqFISH). In this review, we have established a comprehensive classification framework based on the fundamental principles of probe design and the mechanisms of signal amplification. We conducted an in-depth comparison of the strengths and limitations of in situ visualization methodologies in terms of spatial resolution, target throughput, and clinical applications. Furthermore, we explored potential major challenges in the development of in situ nucleic acid visualization technologies and proposed potential optimization strategies to address them. Finally, we looked ahead to the future directions for the expansion and development of in situ nucleic acid visualization technologies and analyzed their unique value in clinical translation.