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...

Descripción completa

Detalles Bibliográficos
Autores principales: Meijering, Anna E C, Biebricher, Andreas S, Sitters, Gerrit, Brouwer, Ineke, Peterman, Erwin J G, Wuite, Gijs J L, Heller, Iddo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
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
_version_ 1783511956128268288
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
work_keys_str_mv AT meijeringannaec imagingunlabeledproteinsondnawithsuperresolution
AT biebricherandreass imagingunlabeledproteinsondnawithsuperresolution
AT sittersgerrit imagingunlabeledproteinsondnawithsuperresolution
AT brouwerineke imagingunlabeledproteinsondnawithsuperresolution
AT petermanerwinjg imagingunlabeledproteinsondnawithsuperresolution
AT wuitegijsjl imagingunlabeledproteinsondnawithsuperresolution
AT helleriddo imagingunlabeledproteinsondnawithsuperresolution