ArticleNucleic acids research2018
Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
- Applications and development of in situ nucleic acid visualization techniques.Frontiers in bioengineering and biotechnology · 2026Review
- Electrochemically modulated single-molecule localization microscopy forNanophotonics (Berlin, Germany) · 2025Article
- Imaging of endogenous RNA in live cells using sequence-activated fluorescent RNA probes.Nucleic acids research · 2025Article
- Quantitative multiplexed analysis of gene and protein expression patterns inCurrent research in microbial sciences · 2025Article
- Recent Advances in Photoswitchable Fluorescent and Colorimetric Probes.Molecules (Basel, Switzerland) · 2024Review
- Regulation of β-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.Cell communication and signaling : CCS · 2024Article
- β-Cell Heterogeneity and Plasticity.Advances in anatomy, embryology, and cell biology · 2024Review
- Expansion microscopy using a single anchor molecule for high-yield multiplexed imaging of proteins and RNAs.PloS one · 2023Article
- Probing β-Cell Biology in Space and Time.Diabetes · 2021Review
- Estimating the dynamic range of quantitative single-molecule localization microscopy.Biophysical journal · 2021Article
- Multiscale plant modeling: from genome to phenome and beyond.Emerging topics in life sciences · 2021Review
- Single Molecule-Based fliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet β-Cells.Diabetes · 2021Article
- An expectation-maximization approach to quantifying protein stoichiometry with single-molecule imaging.Bioinformatics advances · 2021Article
- SP1-upregulated LBX2-AS1 promotes the progression of glioma by targeting the miR-491-5p/LIF axis.Journal of Cancer · 2021Article
- Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy.Disease models & mechanisms · 2020Review
- Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis.Molecular cancer · 2020Article
- Article
- The Long Noncoding RNA Paupar Modulates PAX6 Regulatory Activities to Promote Alpha Cell Development and Function.Cell metabolism · 2019Article
- Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes.Diabetes · 2019Article
- Modelling the endocrine pancreas in health and disease.Nature reviews. Endocrinology · 2019Review
Corrections and comments
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Authors and funding
8 authors.
Funding
Abstract
Quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.
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