Protocol Optimization And Performance Evaluation Of Hybridization Chain Reaction Fluorescence In Situ Hybridization(HCR-FISH)

Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the microbes in environmental samples such as marine sediment have limited cell size and activity,and the number of their ribosomal RNA as well.Therefore,the usage of r RNA targeted traditional FISH is restricted for its low signal intensity on these microbes.On the other hand,the various abiotic particles in marine sediment induce severe false positive signal issue.Usually,the signal intensity can be improved by integrating signal amplification process,while the false-positive signal can be reduced by the optimization of FISH protocol and the application of proper counterstaining method.In this study,we attempted to validate and optimize FISH on sediment samples by integrating Hybridization Chain Reaction(HCR),a signal amplification process.HCR indicates a production of long DNA chain from a repeating hybridization of two species of DNA hairpins.The process requires an oligo-DNA as trigger but doesn't require any enzyme or change of temperature.Focus on HCR-FISH,this study verified a series of methods to reduce false-positive signal to find an optimal HCR-FISH protocol that fits sediment samples.Its performance was also evaluated quantitatively.In this study,the performance of original HCR-FISH protocol was tested on laboratory cultured bacterium Escherichia coli,where we found that increasing the concentration of initiator probe in hybridization buffer is key to improve the robustness of HCR-FISH.The optimized HCR-FISH protocol was validated on laboratory cultured archaea Methanococcoides methylutens and Thermococcus eurythermalis.Five sets of orthogonal HCR probe were tested and two sets were selected for further study owing to their robustness and efficiency.To lower the impact of abiotic particles on the performance of HCR-FISH,several cell detachment and extraction methods were tested and one efficient method was selected;three types of hybridization buffers were evaluated on their abilities to repress false-positive signal;the difference on the fluorescence spectra of DAPI-DNA complex and DAPI-abiotic particle complex was discovered,and an image-processing method was come up with based on the discovery to reduce the false-positive signal on counterstaining images;two different types of counterstaining reagent were tested and their protocols were also documented.In the end,a standard protocol of HCR-FISH for sediment samples was summarized and tested on sediment samples from South China Sea.Its performance was also evaluated quantitatively compared with traditional FISH.HCR-FISH,as a relatively easy using signal-enhanced hybridization technology,can serve as an important tool to study uncultured microbes in environment.This study proposed a robust HCR-FISH protocol on sediment samples,and may work well on seawater and fresh water samples as well.The research on counterstaining provided a useful tool for other microscopic researches to differentiate cells and abiotic particles.The optimized protocol of HCR-FISH proposed in this study would provide essential help on research on uncultured environmental microorganism.