Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

Abstract

Here, 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 RNAmore » copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [2];  [1]
+ Show Author Affiliations
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  2. Univ. of Colorado Denver, Aurora, CO (United States)
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1435646
Report Number(s):
PNNL-SA-126413
Journal ID: ISSN 0305-1048
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 46; Journal Issue: 2; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, and Orr, Galya. Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells. United States: N. p., 2017. Web. doi:10.1093/nar/gkx874.
Copy to clipboard
Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, & Orr, Galya. Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells. United States. https://doi.org/10.1093/nar/gkx874
Copy to clipboard
Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, and Orr, Galya. Wed . "Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells". United States. https://doi.org/10.1093/nar/gkx874. https://www.osti.gov/servlets/purl/1435646.
Copy to clipboard
@article{osti_1435646,
title = {Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells},
author = {Cui, Yi and Hu, Dehong and Markillie, Lye Meng and Chrisler, William B. and Gaffrey, Matthew J. and Ansong, Charles and Sussel, Lori and Orr, Galya},
abstractNote = {Here, 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.},
doi = {10.1093/nar/gkx874},
journal = {Nucleic Acids Research},
number = 2,
volume = 46,
place = {United States},
year = {Wed Oct 04 00:00:00 EDT 2017},
month = {Wed Oct 04 00:00:00 EDT 2017}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1093/nar/gkx874
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 22 works
Citation information provided by
Web of Science
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Super-Resolution Imaging with Small Organic Fluorophores
journal, September 2009

  • Heilemann, Mike; van de Linde, Sebastian; Mukherjee, Anindita
  • Angewandte Chemie International Edition, Vol. 48, Issue 37
  • DOI: 10.1002/anie.200902073

Stochastic protein expression in individual cells at the single molecule level
journal, March 2006

  • Cai, Long; Friedman, Nir; Xie, X. Sunney
  • Nature, Vol. 440, Issue 7082
  • DOI: 10.1038/nature04599

Nkx2.2 Regulates β-Cell Function in the Mature Islet
journal, April 2007

Single-Cell Gene Expression Profiling
journal, August 2002

Single-molecule mRNA detection and counting in mammalian tissue
journal, August 2013

  • Lyubimova, Anna; Itzkovitz, Shalev; Junker, Jan Philipp
  • Nature Protocols, Vol. 8, Issue 9
  • DOI: 10.1038/nprot.2013.109

Spatially resolved, highly multiplexed RNA profiling in single cells
journal, April 2015

Visualization of Single RNA Transcripts in Situ
journal, April 1998

Stochastic mRNA Synthesis in Mammalian Cells
journal, September 2006

Imaging Intracellular Fluorescent Proteins at Nanometer Resolution
journal, September 2006

Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami
journal, November 2010

  • Jungmann, Ralf; Steinhauer, Christian; Scheible, Max
  • Nano Letters, Vol. 10, Issue 11
  • DOI: 10.1021/nl103427w

In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus
journal, October 2016

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy
journal, May 2009

  • Vogelsang, J.; Cordes, T.; Forthmann, C.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 20
  • DOI: 10.1073/pnas.0811875106

Integrated genome and transcriptome sequencing of the same cell
journal, January 2015

  • Dey, Siddharth S.; Kester, Lennart; Spanjaard, Bastiaan
  • Nature Biotechnology, Vol. 33, Issue 3
  • DOI: 10.1038/nbt.3129

Transcription and translation initiation frequencies of the Escherichia coli lac operon
journal, July 1977

Direct stochastic optical reconstruction microscopy with standard fluorescent probes
journal, June 2011

  • van de Linde, Sebastian; Löschberger, Anna; Klein, Teresa
  • Nature Protocols, Vol. 6, Issue 7
  • DOI: 10.1038/nprot.2011.336

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
journal, September 2016

  • Moffitt, Jeffrey R.; Hao, Junjie; Wang, Guiping
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 39
  • DOI: 10.1073/pnas.1612826113

Single-cell in situ RNA profiling by sequential hybridization
journal, March 2014

  • Lubeck, Eric; Coskun, Ahmet F.; Zhiyentayev, Timur
  • Nature Methods, Vol. 11, Issue 4
  • DOI: 10.1038/nmeth.2892

Quantitative super-resolution imaging with qPAINT
journal, March 2016

  • Jungmann, Ralf; Avendaño, Maier S.; Dai, Mingjie
  • Nature Methods, Vol. 13, Issue 5
  • DOI: 10.1038/nmeth.3804

Inside single cells: quantitative analysis with advanced optics and nanomaterials
journal, November 2014

  • Cui, Yi; Irudayaraj, Joseph
  • Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, Vol. 7, Issue 3
  • DOI: 10.1002/wnan.1321

Single-cell spatial reconstruction reveals global division of labour in the mammalian liver
journal, February 2017

  • Halpern, Keren Bahar; Shenhav, Rom; Matcovitch-Natan, Orit
  • Nature, Vol. 542, Issue 7641
  • DOI: 10.1038/nature21065

Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT
journal, February 2014

  • Jungmann, Ralf; Avendaño, Maier S.; Woehrstein, Johannes B.
  • Nature Methods, Vol. 11, Issue 3
  • DOI: 10.1038/nmeth.2835

Super-Resolution Fluorescence Microscopy
journal, June 2009

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging
journal, November 2011

  • Dempsey, Graham T.; Vaughan, Joshua C.; Chen, Kok Hao
  • Nature Methods, Vol. 8, Issue 12
  • DOI: 10.1038/nmeth.1768

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing
journal, November 2016

  • Moffitt, Jeffrey R.; Hao, Junjie; Bambah-Mukku, Dhananjay
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 50
  • DOI: 10.1073/pnas.1617699113

Impact of islet architecture on β-cell heterogeneity, plasticity and function
journal, September 2016

  • Roscioni, Sara S.; Migliorini, Adriana; Gegg, Moritz
  • Nature Reviews Endocrinology, Vol. 12, Issue 12
  • DOI: 10.1038/nrendo.2016.147

Beyond quantification: in situ analysis of transcriptome and pre-mRNA alternative splicing at the nanoscale
journal, November 2016

  • Cui, Yi; Liu, Jing; Irudayaraj, Joseph
  • Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, Vol. 9, Issue 4
  • DOI: 10.1002/wnan.1443

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
journal, August 2006

  • Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei
  • Nature Methods, Vol. 3, Issue 10
  • DOI: 10.1038/nmeth929

Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution
journal, February 2016

  • Sylwestrak, Emily Lauren; Rajasethupathy, Priyamvada; Wright, Matthew Arnot
  • Cell, Vol. 164, Issue 4
  • DOI: 10.1016/j.cell.2016.01.038

smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability
journal, September 2016

  • Tsanov, Nikolay; Samacoits, Aubin; Chouaib, Racha
  • Nucleic Acids Research, Vol. 44, Issue 22
  • DOI: 10.1093/nar/gkw784

Identification of proliferative and mature β-cells in the islets of Langerhans
journal, July 2016

  • Bader, Erik; Migliorini, Adriana; Gegg, Moritz
  • Nature, Vol. 535, Issue 7612
  • DOI: 10.1038/nature18624

Single-cell systems biology by super-resolution imaging and combinatorial labeling
journal, June 2012

Optical Clearing Delivers Ultrasensitive Hyperspectral Dark-Field Imaging for Single-Cell Evaluation
journal, February 2016

Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq
journal, October 2016

  • Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.
  • ACS Nano, Vol. 10, Issue 11
  • DOI: 10.1021/acsnano.6b05452

Human islets contain four distinct subtypes of β cells
journal, July 2016

  • Dorrell, Craig; Schug, Jonathan; Canaday, Pamela S.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms11756

Nature, Nurture, or Chance: Stochastic Gene Expression and Its Consequences
journal, October 2008

Imaging individual mRNA molecules using multiple singly labeled probes
journal, September 2008

  • Raj, Arjun; van den Bogaard, Patrick; Rifkin, Scott A.
  • Nature Methods, Vol. 5, Issue 10
  • DOI: 10.1038/nmeth.1253

Validating transcripts with probes and imaging technology
journal, March 2011

  • Itzkovitz, Shalev; van Oudenaarden, Alexander
  • Nature Methods, Vol. 8, Issue S4
  • DOI: 10.1038/nmeth.1573

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging
journal, January 2016

  • Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan
  • Biomedical Optics Express, Vol. 7, Issue 3
  • DOI: 10.1364/BOE.7.000855

Single-Cell Mass Cytometry Analysis of the Human Endocrine Pancreas
journal, October 2016

Nanoscale imaging of RNA with expansion microscopy
journal, July 2016

  • Chen, Fei; Wassie, Asmamaw T.; Cote, Allison J.
  • Nature Methods, Vol. 13, Issue 8
  • DOI: 10.1038/nmeth.3899

Quantitative mRNA imaging throughout the entire Drosophila brain
journal, June 2017

  • Long, Xi; Colonell, Jennifer; Wong, Allan M.
  • Nature Methods, Vol. 14, Issue 7
  • DOI: 10.1038/nmeth.4309

Optical imaging of individual biomolecules in densely packed clusters
journal, July 2016

  • Dai, Mingjie; Jungmann, Ralf; Yin, Peng
  • Nature Nanotechnology, Vol. 11, Issue 9
  • DOI: 10.1038/nnano.2016.95

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy
journal, December 2012

  • Abrahamsson, Sara; Chen, Jiji; Hajj, Bassam
  • Nature Methods, Vol. 10, Issue 1
  • DOI: 10.1038/nmeth.2277

Inside single cells: quantitative analysis with advanced optics and nanomaterials
journal, November 2014

  • Cui, Yi; Irudayaraj, Joseph
  • Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, Vol. 7, Issue 3
  • DOI: 10.1002/wnan.1321

Transcription and translation initiation frequencies of the Escherichia coli lac operon
journal, July 1977

Nature, Nurture, or Chance: Stochastic Gene Expression and Its Consequences
journal, October 2008

Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution
journal, February 2016

  • Sylwestrak, Emily Lauren; Rajasethupathy, Priyamvada; Wright, Matthew Arnot
  • Cell, Vol. 164, Issue 4
  • DOI: 10.1016/j.cell.2016.01.038

Single-Cell Mass Cytometry Analysis of the Human Endocrine Pancreas
journal, October 2016

In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus
journal, October 2016

Precise Nanometer Localization Analysis for Individual Fluorescent Probes
journal, May 2002

Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq
journal, October 2016

  • Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.
  • ACS Nano, Vol. 10, Issue 11
  • DOI: 10.1021/acsnano.6b05452

Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami
journal, November 2010

  • Jungmann, Ralf; Steinhauer, Christian; Scheible, Max
  • Nano Letters, Vol. 10, Issue 11
  • DOI: 10.1021/nl103427w

Stochastic protein expression in individual cells at the single molecule level
journal, March 2006

  • Cai, Long; Friedman, Nir; Xie, X. Sunney
  • Nature, Vol. 440, Issue 7082
  • DOI: 10.1038/nature04599

Integrated genome and transcriptome sequencing of the same cell
journal, January 2015

  • Dey, Siddharth S.; Kester, Lennart; Spanjaard, Bastiaan
  • Nature Biotechnology, Vol. 33, Issue 3
  • DOI: 10.1038/nbt.3129

Single-molecule transcript counting of stem-cell markers in the mouse intestine
journal, November 2011

  • Itzkovitz, Shalev; Lyubimova, Anna; Blat, Irene C.
  • Nature Cell Biology, Vol. 14, Issue 1
  • DOI: 10.1038/ncb2384

Imaging individual mRNA molecules using multiple singly labeled probes
journal, September 2008

  • Raj, Arjun; van den Bogaard, Patrick; Rifkin, Scott A.
  • Nature Methods, Vol. 5, Issue 10
  • DOI: 10.1038/nmeth.1253

Validating transcripts with probes and imaging technology
journal, March 2011

  • Itzkovitz, Shalev; van Oudenaarden, Alexander
  • Nature Methods, Vol. 8, Issue S4
  • DOI: 10.1038/nmeth.1573

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging
journal, November 2011

  • Dempsey, Graham T.; Vaughan, Joshua C.; Chen, Kok Hao
  • Nature Methods, Vol. 8, Issue 12
  • DOI: 10.1038/nmeth.1768

Single-cell systems biology by super-resolution imaging and combinatorial labeling
journal, June 2012

Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT
journal, February 2014

  • Jungmann, Ralf; Avendaño, Maier S.; Woehrstein, Johannes B.
  • Nature Methods, Vol. 11, Issue 3
  • DOI: 10.1038/nmeth.2835

Highly multiplexed transcriptome imaging
journal, May 2015

Quantitative super-resolution imaging with qPAINT
journal, March 2016

  • Jungmann, Ralf; Avendaño, Maier S.; Dai, Mingjie
  • Nature Methods, Vol. 13, Issue 5
  • DOI: 10.1038/nmeth.3804

The quest for multiplexed spatially resolved transcriptional profiling
journal, July 2016

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
journal, August 2006

  • Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei
  • Nature Methods, Vol. 3, Issue 10
  • DOI: 10.1038/nmeth929

Optical imaging of individual biomolecules in densely packed clusters
journal, July 2016

  • Dai, Mingjie; Jungmann, Ralf; Yin, Peng
  • Nature Nanotechnology, Vol. 11, Issue 9
  • DOI: 10.1038/nnano.2016.95

Single-molecule mRNA detection and counting in mammalian tissue
journal, August 2013

  • Lyubimova, Anna; Itzkovitz, Shalev; Junker, Jan Philipp
  • Nature Protocols, Vol. 8, Issue 9
  • DOI: 10.1038/nprot.2013.109

Impact of islet architecture on β-cell heterogeneity, plasticity and function
journal, September 2016

  • Roscioni, Sara S.; Migliorini, Adriana; Gegg, Moritz
  • Nature Reviews Endocrinology, Vol. 12, Issue 12
  • DOI: 10.1038/nrendo.2016.147

The limits of light
journal, September 2010

  • Schuldt, Alison
  • Nature Reviews Molecular Cell Biology, Vol. 11, Issue 10
  • DOI: 10.1038/nrm2989

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
journal, September 2016

  • Moffitt, Jeffrey R.; Hao, Junjie; Wang, Guiping
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 39
  • DOI: 10.1073/pnas.1612826113

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing
journal, November 2016

  • Moffitt, Jeffrey R.; Hao, Junjie; Bambah-Mukku, Dhananjay
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 50
  • DOI: 10.1073/pnas.1617699113

smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability
journal, September 2016

  • Tsanov, Nikolay; Samacoits, Aubin; Chouaib, Racha
  • Nucleic Acids Research, Vol. 44, Issue 22
  • DOI: 10.1093/nar/gkw784

Single-Cell Gene Expression Profiling
journal, August 2002

Visualization of Single RNA Transcripts in Situ
journal, April 1998

Pancreatic β cell identity requires continual repression of non–β cell programs
journal, December 2016

  • Gutiérrez, Giselle Domínguez; Bender, Aaron S.; Cirulli, Vincenzo
  • Journal of Clinical Investigation, Vol. 127, Issue 1
  • DOI: 10.1172/jci88017

Heterogeneity of the Pancreatic Beta Cell
journal, March 2017

  • Gutierrez, Giselle Dominguez; Gromada, Jesper; Sussel, Lori
  • Frontiers in Genetics, Vol. 8
  • DOI: 10.3389/fgene.2017.00022

Genetic evidence that Nkx2.2 acts primarily downstream of Neurog3 in pancreatic endocrine lineage development
journal, January 2017

  • Churchill, Angela J.; Gutiérrez, Giselle Dominguez; Singer, Ruth A.
  • eLife, Vol. 6
  • DOI: 10.7554/elife.20010
    Sort by:
    [ × clear filter / sort ]

    Works referencing / citing this record:

    Genetic and metabolic links between the murine microbiome and memory
    journal, April 2020

    New Understanding of β-Cell Heterogeneity and In Situ Islet Function
    journal, March 2018

    • Benninger, Richard K. P.; Hodson, David J.
    • Diabetes, Vol. 67, Issue 4
    • DOI: 10.2337/dbi17-0040
      Sort by:
      [ × clear filter / sort ]

      Similar Records in DOE PAGES and OSTI.GOV collections: