Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect singlemolecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.
Bibliographical noteFunding Information:
This work was supported by the Heritage Medical Research Institute [HMRI-15-09-01 to V.G.]; the National Institutes of Health [1DP2OD017782 to V.G.; 5R01EB006192 to N.A.P.; R01 HD075605 and 1DP2OD008530 to L.C.]; the Beckman Institute at Caltech [PMTC, CLOVER (http://www.beckmaninstitute. caltech.edu/clover.shtml) and pilot center to L.C.]; a Caltech Amgen Chem-Bio-Engineering award [CBEA, grant 7200811262]; the Gordon and Betty Moore Foundation [GBMF2809]; the National Science Foundation Molecular Programming Project [NSF-CCF-1317694]; the John Simon Guggenheim Memorial Foundation (N.A.P.); and the McKnight Foundation (L.C.). Deposited in PMC for immediate release.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Developmental Biology