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Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.

Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT - Nat Commun (2015)

Bottom Line: We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems.Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals.These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

No MeSH data available.


Related in: MedlinePlus

Super-resolution imaging with Oligopaints and DNA-PAINT.(a) Labelling scheme using Oligopaint probes carrying an ATTO 488 dye and a 9-base docking site that is complementary to imager strands labelled with ATTO 655. (b) Trace of Intensity versus time showing the transient binding of imager strands and docking strands or ‘blinks'. (c,d) Diffraction-limited images obtained with ATTO 488 (left) and DNA-PAINT super-resolution images obtained with ATTO 655-labelled imager strands at 5 nM (right) of Oligopaint probe sets labelled with ATTO 488 and targeting 174 kb (c) and 5 kb (d) of the mouse hoxB locus in MEFs. To the right of the images are cross-sectional (dotted lines in DNA-PAINT images i–ix) histograms displaying the normalized number of photons detected (normalized counts) versus transverse position for each region. Structural features are inferred from these transverses with one-dimensional Gaussian fits, with FWHMs indicated above each graph. Imaging: 15,000 frames at 10 Hz rate. Super-resolution images are presented as heat maps of single-mole localization density: black (fewest) -> red -> yellow -> white (most).
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f3: Super-resolution imaging with Oligopaints and DNA-PAINT.(a) Labelling scheme using Oligopaint probes carrying an ATTO 488 dye and a 9-base docking site that is complementary to imager strands labelled with ATTO 655. (b) Trace of Intensity versus time showing the transient binding of imager strands and docking strands or ‘blinks'. (c,d) Diffraction-limited images obtained with ATTO 488 (left) and DNA-PAINT super-resolution images obtained with ATTO 655-labelled imager strands at 5 nM (right) of Oligopaint probe sets labelled with ATTO 488 and targeting 174 kb (c) and 5 kb (d) of the mouse hoxB locus in MEFs. To the right of the images are cross-sectional (dotted lines in DNA-PAINT images i–ix) histograms displaying the normalized number of photons detected (normalized counts) versus transverse position for each region. Structural features are inferred from these transverses with one-dimensional Gaussian fits, with FWHMs indicated above each graph. Imaging: 15,000 frames at 10 Hz rate. Super-resolution images are presented as heat maps of single-mole localization density: black (fewest) -> red -> yellow -> white (most).

Mentions: We also explored the potential of Oligopaint primary–secondary pairings to enable the visualization of single-copy genomic regions using a related single-molecule-based super-resolution approach called DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT)363738. In DNA-PAINT, the single-molecule fluorescence events are generated by the transient hybridization of fluorescently labelled oligonucleotides, called ‘imager strands', present in solution in the imaging buffer to complementary strands, called ‘docking strands', on the target to be imaged, reminiscent of the binding of secondary oligos to the MainStreet of Oligopaints (Fig. 3a); as the duplexes that form are designed to be unstable at room temperature (RT, duplex length of 9 bases; bound time in imaging conditions ≈600 ms (ref. 37), the transient binding interactions lead to an apparent ‘blinking' of the docking sites when imaged using configurations, such as total internal reflection fluorescence (TIRF) microscopy or highly inclined and laminated optical sheet39 microscopy, which provide high ratios of signal:noise (Fig. 3b).


Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.

Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT - Nat Commun (2015)

Super-resolution imaging with Oligopaints and DNA-PAINT.(a) Labelling scheme using Oligopaint probes carrying an ATTO 488 dye and a 9-base docking site that is complementary to imager strands labelled with ATTO 655. (b) Trace of Intensity versus time showing the transient binding of imager strands and docking strands or ‘blinks'. (c,d) Diffraction-limited images obtained with ATTO 488 (left) and DNA-PAINT super-resolution images obtained with ATTO 655-labelled imager strands at 5 nM (right) of Oligopaint probe sets labelled with ATTO 488 and targeting 174 kb (c) and 5 kb (d) of the mouse hoxB locus in MEFs. To the right of the images are cross-sectional (dotted lines in DNA-PAINT images i–ix) histograms displaying the normalized number of photons detected (normalized counts) versus transverse position for each region. Structural features are inferred from these transverses with one-dimensional Gaussian fits, with FWHMs indicated above each graph. Imaging: 15,000 frames at 10 Hz rate. Super-resolution images are presented as heat maps of single-mole localization density: black (fewest) -> red -> yellow -> white (most).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430122&req=5

f3: Super-resolution imaging with Oligopaints and DNA-PAINT.(a) Labelling scheme using Oligopaint probes carrying an ATTO 488 dye and a 9-base docking site that is complementary to imager strands labelled with ATTO 655. (b) Trace of Intensity versus time showing the transient binding of imager strands and docking strands or ‘blinks'. (c,d) Diffraction-limited images obtained with ATTO 488 (left) and DNA-PAINT super-resolution images obtained with ATTO 655-labelled imager strands at 5 nM (right) of Oligopaint probe sets labelled with ATTO 488 and targeting 174 kb (c) and 5 kb (d) of the mouse hoxB locus in MEFs. To the right of the images are cross-sectional (dotted lines in DNA-PAINT images i–ix) histograms displaying the normalized number of photons detected (normalized counts) versus transverse position for each region. Structural features are inferred from these transverses with one-dimensional Gaussian fits, with FWHMs indicated above each graph. Imaging: 15,000 frames at 10 Hz rate. Super-resolution images are presented as heat maps of single-mole localization density: black (fewest) -> red -> yellow -> white (most).
Mentions: We also explored the potential of Oligopaint primary–secondary pairings to enable the visualization of single-copy genomic regions using a related single-molecule-based super-resolution approach called DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT)363738. In DNA-PAINT, the single-molecule fluorescence events are generated by the transient hybridization of fluorescently labelled oligonucleotides, called ‘imager strands', present in solution in the imaging buffer to complementary strands, called ‘docking strands', on the target to be imaged, reminiscent of the binding of secondary oligos to the MainStreet of Oligopaints (Fig. 3a); as the duplexes that form are designed to be unstable at room temperature (RT, duplex length of 9 bases; bound time in imaging conditions ≈600 ms (ref. 37), the transient binding interactions lead to an apparent ‘blinking' of the docking sites when imaged using configurations, such as total internal reflection fluorescence (TIRF) microscopy or highly inclined and laminated optical sheet39 microscopy, which provide high ratios of signal:noise (Fig. 3b).

Bottom Line: We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems.Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals.These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

No MeSH data available.


Related in: MedlinePlus