Imaging unlabeled proteins on DNA with super-resolution
Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce invers...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102996/ https://www.ncbi.nlm.nih.gov/pubmed/32016413 http://dx.doi.org/10.1093/nar/gkaa061 |
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author | Meijering, Anna E C Biebricher, Andreas S Sitters, Gerrit Brouwer, Ineke Peterman, Erwin J G Wuite, Gijs J L Heller, Iddo |
author_facet | Meijering, Anna E C Biebricher, Andreas S Sitters, Gerrit Brouwer, Ineke Peterman, Erwin J G Wuite, Gijs J L Heller, Iddo |
author_sort | Meijering, Anna E C |
collection | PubMed |
description | Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling. |
format | Online Article Text |
id | pubmed-7102996 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-71029962020-04-02 Imaging unlabeled proteins on DNA with super-resolution Meijering, Anna E C Biebricher, Andreas S Sitters, Gerrit Brouwer, Ineke Peterman, Erwin J G Wuite, Gijs J L Heller, Iddo Nucleic Acids Res Methods Online Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling. Oxford University Press 2020-04-06 2020-02-04 /pmc/articles/PMC7102996/ /pubmed/32016413 http://dx.doi.org/10.1093/nar/gkaa061 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Meijering, Anna E C Biebricher, Andreas S Sitters, Gerrit Brouwer, Ineke Peterman, Erwin J G Wuite, Gijs J L Heller, Iddo Imaging unlabeled proteins on DNA with super-resolution |
title | Imaging unlabeled proteins on DNA with super-resolution |
title_full | Imaging unlabeled proteins on DNA with super-resolution |
title_fullStr | Imaging unlabeled proteins on DNA with super-resolution |
title_full_unstemmed | Imaging unlabeled proteins on DNA with super-resolution |
title_short | Imaging unlabeled proteins on DNA with super-resolution |
title_sort | imaging unlabeled proteins on dna with super-resolution |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102996/ https://www.ncbi.nlm.nih.gov/pubmed/32016413 http://dx.doi.org/10.1093/nar/gkaa061 |
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