Abstract

Effect of different organic fluorophores on signal quality and multiplexing ability of fluorescence in situ hybridization chain reaction method

Kumar V, Krolweski DM, Parsegian A, Foltz M, Martin B, Akil H, Watson SJ
50th Annual Meeting of the Society for Neuroscience, Virtual. 2021.

Abstract

Fluorescence in situ hybridization chain reaction (HCR FISH) offers one of the most efficient and simple methods of signal amplification for transcript probing in varied technological settings including thin section as well as large intact tissues (Choi et. al., 2014, 2018, Sylwestrak et. al., 2016, Kumar et. al., 2021). Applying multiple custom designed split-pair probes, and fluorophore-labeled DNA hairpins to tissue concurrently allows for multiplexing of up to five targets with high specificity. Consistent fluorescence signal with optimum signal to noise (S/N) ratio is a determining factor for both qualitative and quantitative analysis, especially dependent upon the colocalization. Through our repeated studies using thin sections (10-30 µm) as well as iDISCO+ processed large rodent brain tissues (few mm thick slices to intact hemispheres), we discovered that effectiveness of the HCR FISH technique was critically dependent on the choice of organic dyes used as fluorophores. For example, we consistently found that hairpins conjugated with AlexaFluor-647 (AF-647) produced the most reliable fluorescence signal with highest S/N ratio and proved to be the most resilient to buffers and mounting mediums as well as photobleaching under extended imaging sessions using both confocal and light sheet microscopes. In contrast, hairpins tagged with AF-405/-488/-594 often failed to produce as consistent and comparable S/N ratios across the experiments, however it was dependent upon the target gene and tissue processing method. The fluorophore-based differences were most evident when HCR FISH was performed on low copy number genes using perfused and or iDISCO+ cleared brain samples. Next, we tested DNA hairpins conjugated with non-AF fluorophores, FITC, Cy3, JaneliaFluor-525 and BODIPY-590, but these dyes too failed to produce a comparable signal to that of AF-647. Here, we report and discuss above-mentioned findings and their potential mechanisms. Then, we explore the future directions to establish the exact cause of such fluorophore-based differences in signal output and alternate solutions to achieve adequate and consistent fluorescence for multiplexed labeling and benefit both qualitative as well as quantitative studies.