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Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?
Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to dete...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516992/ https://www.ncbi.nlm.nih.gov/pubmed/28723958 http://dx.doi.org/10.1371/journal.pone.0180871 |
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author | Mollazade, Mahdie Tabarin, Thibault Nicovich, Philip R. Soeriyadi, Alexander Nieves, Daniel J. Gooding, J. Justin Gaus, Katharina |
author_facet | Mollazade, Mahdie Tabarin, Thibault Nicovich, Philip R. Soeriyadi, Alexander Nieves, Daniel J. Gooding, J. Justin Gaus, Katharina |
author_sort | Mollazade, Mahdie |
collection | PubMed |
description | Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision. |
format | Online Article Text |
id | pubmed-5516992 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-55169922017-08-07 Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? Mollazade, Mahdie Tabarin, Thibault Nicovich, Philip R. Soeriyadi, Alexander Nieves, Daniel J. Gooding, J. Justin Gaus, Katharina PLoS One Research Article Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision. Public Library of Science 2017-07-19 /pmc/articles/PMC5516992/ /pubmed/28723958 http://dx.doi.org/10.1371/journal.pone.0180871 Text en © 2017 Mollazade et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Mollazade, Mahdie Tabarin, Thibault Nicovich, Philip R. Soeriyadi, Alexander Nieves, Daniel J. Gooding, J. Justin Gaus, Katharina Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title | Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title_full | Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title_fullStr | Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title_full_unstemmed | Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title_short | Can single molecule localization microscopy be used to map closely spaced RGD nanodomains? |
title_sort | can single molecule localization microscopy be used to map closely spaced rgd nanodomains? |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516992/ https://www.ncbi.nlm.nih.gov/pubmed/28723958 http://dx.doi.org/10.1371/journal.pone.0180871 |
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