A starter kit for point-localization super-resolution imaging

Manley, Suliana; Gunzenhäuser, Julia; Olivier, Nicolas

doi:10.1016/j.cbpa.2011.10.009

Cited by 22 publications

(18 citation statements)

References 66 publications

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“…To more directly examine the conformation of the HoxD cluster in single cells, we labeled its entire genomic sequence by using a 160-kb large BAC as a DNA probe and carried out DNA FISH. The morphology of the HoxD cluster was subsequently resolved using stochastic optical reconstruction microscopy (STORM), a type of super-resolution imaging based on sequential imaging of single-molecule signals (24)(25)(26). As a control, we used a probe of similar size located nearby, within the telomeric gene desert ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

Fabre

Benke

Joye

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context. DNA FISH | super-resolution microscopy | limb development | topologically associating domains | HoxD

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Section: Resultsmentioning

confidence: 99%

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

Fabre

Benke

Joye

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…When no commercial setup is available, microscopy setups equipped with high-power excitation lasers (≤100 mW; power for individual lasers given in MATERIALS section), high-sensitivity cameras (e.g., electron-multiplying charge-coupled device (EM-CCD) cameras), a high-numerical-aperture (high-NA) objective (NA ≥ 1.45) and z-drift correction for single-molecule imaging should be used instead 43 . Users who are not experts in the field of single-molecule imaging should seek advice from an expert in the field on issues such as laser safety precautions and adjusting settings, such as the TIRF mode.…”

Section: Superresolution Microscopy Setup and Data Analysismentioning

confidence: 99%

“…AF647 is activated into the fluorescent on-state by the 405-nm laser line and, if the microscopy set up is equipped with that feature, the 405-nm laser power will be automatically adjusted to keep the number of molecules in the fluorescent state low and constant during the acquisition. Detailed protocols and troubleshooting for the imaging process are available elsewhere 15,43,50 .  crItIcal step When you are mounting the sample holder on the stage, clean the bottom of the coverslip thoroughly with 70% (vol/vol) ethanol, always wiping in the same direction and carefully drying it afterward.…”

Section: (B) If Using Bsa Gs or Hsmentioning

confidence: 99%

Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast

Kaplan

Ewers

2015

Nat Protoc

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Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2-4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.

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“…STORM involves the sequential blinking and localization of single-molecule signals to reconstruct superresolution images ( Fig. 3D; Huang et al 2008;Manley et al 2011;Benke and Manley 2012). The control was a BAC of similar size located nearby in the telomeric gene desert (Fig.…”

Section: The Hoxd Locus At Superresolutionmentioning

confidence: 99%

Visualizing theHoxDGene Cluster at the Nanoscale Level

Fabre

Benke

Manley

et al. 2015

Cold Spring Harb Symp Quant Biol

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Transcription of HoxD cluster genes in limbs is coordinated by two topologically associating domains (TADs), neighboring the cluster and containing various enhancers. Here, we use a combination of microscopy approaches and chromosome conformation capture to assess the structural changes occurring in this global architecture in various functional states. We observed that despite their spatial juxtaposition, the TADs are consistently kept as distinct three-dimensional units. Hox genes located at their boundary can show significant spatial segregation over long distances, suggesting that physical elongation of the HoxD cluster occurs. The use of superresolution imaging (STORM [stochastic optical reconstruction microscopy]) revealed that the gene cluster can be in an either compact or elongated shape. The latter configuration is observed in transcriptionally active tissue and in embryonic stem cells, consistent with chromosome conformation capture results. Such morphological changes at HoxD in developing digits seem to be associated with its position at the boundary between two TADs and support the idea that chromatin dynamics is important in the establishment of transcriptional activity.

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A starter kit for point-localization super-resolution imaging

Cited by 22 publications

References 66 publications

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast

Visualizing theHoxDGene Cluster at the Nanoscale Level

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