A new technique called split-FISH allows researchers to analyze over 300 genes within intact tissue at the same time.

© Genome Institute of Singapore

FISHing out tissues’ hidden secrets

14 Apr 2021

A*STAR researchers have developed a new molecular imaging technique that enables the study of tissue physiology at ultra-high resolution.

Like miniature cities, human tissues can be thought of as cellular “buildings” clustered strategically within a three-dimensional space. Just as shops and transit hubs are often situated close to bustling business districts, the physical locations of different cell types hold valuable clues about their roles in both normal and diseased tissues.

To map out intricate tissue structures, scientists typically use a technique called fluorescence in situ hybridization, or FISH. FISH allows researchers to visualize individual RNA molecules within intact tissues by using fluorescent probes that bind to the genes of interest.

“This information has given us insight into the inner workings of a tissue which could accelerate numerous biological research areas, such as neuroscience, developmental biology, immuno-oncology, and host-pathogen interactions,” explained Kok Hao Chen, a Senior Research Scientist at A*STAR’s Genome Institute of Singapore (GIS).

Traditional FISH protocols, however, lack the clarity and resolution required to navigate tissue architectures with precision. Dim signals and background noise are ongoing issues. The multi-colored fluorescent probes are usually applied as concentrated cocktails and often bind to molecules that are not their designated targets, resulting in undesired background fluorescence or even false positives

Now, Chen and a team of scientists have unlocked the next frontier in precision bio-imaging with their newly-developed methodology: split-FISH. This technique features an ingenious in-built mechanism to limit off-target binding. Known as a bridge probe, two independent RNA hybridizations are required to occur in close proximity before the fluorescent beacon is illuminated. The result? Brighter signals and unprecedented accuracy.

“The full process, from tissue processing to imaging, takes about three days,” said Chen, adding that much of the split-FISH workflow is fully automated. “Hybridization of the bridge and readout probes and subsequent imaging steps are controlled by a custom-built, computer-controlled fluidics and imaging system.” The researchers demonstrated the simultaneous visualization of over 300 mouse genes in different tissue types using the platform in a study published in Nature Methods.

Ultimately, imaging 30,000 human genes in concert remains an ambitious target due to the presence of short transcripts which allow fewer fluorescent probes to bind to it, thereby limiting the fluorescent signals from it. By further refining the split-FISH technique, Chen’s group is working on allowing the system to detect these shorter transcripts and look at a greater number of genes. “We also plan to incorporate compatible signal amplification strategies with split-FISH, which will further improve signal quality,” he said.

The A*STAR-affiliated researchers contributing to this research are from the Genome Institute of Singapore (GIS).

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Goh, J.J.L., Chou, N., Seow, W.Y., Ha, N., Cheng, C.P.P., et al. Highly specific multiplexed RNA imaging in tissues with split-FISH. Nature Methods 17, 689–693 (2020) | article

About the Researcher

Kok Hao Chen

Senior Research Scientist

Genome Institute of Singapore
Kok Hao Chen is a Senior Research Scientist at the Genome Institute of Singapore (GIS). He completed his Ph.D. with Xiaowei Zhuang’s group at the Department of Chemistry and Chemical Biology at Harvard University. He graduated with a Bachelor of Science from the Department of Chemical and Biomolecular Engineering with a minor in Physics at the University of Illinois at Urbana-Champaign. He is a recipient of the prestigious AXA Postdoctoral Fellowship Award, NMRC Young Investigator Award, and A*STAR National Science Scholarship.

This article was made for A*STAR Research by Wildtype Media Group