Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes
Publication Date
April 25, 2012
Journal
PLOS ONE
Authors
Christopher J. Tynan, David T. Clarke, Benjamin C. Coles, Daniel J. Rolfe, et al
Volume
7
Issue
4
Pages
e36265
DOI
https://dx.plos.org/10.1371/journal.pone.0036265
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0036265
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/22558412
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338497
Europe PMC
http://europepmc.org/abstract/MED/22558412
Web of Science
000305345200120
Scopus
84865644265
Mendeley
http://www.mendeley.com/research/multicolour-single-molecule-imaging-cells-near-infrared-dyes
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/648253"], "description"=>"<p>The optical properties of selected NIR dyes. Absorption and emission maxima were measured from samples of dyes dissolved in PBS. Molar absorption coefficients, ε<sub>A</sub>, at the peak absorption wavelength were obtained from the manufacturers. The brightness of dyes was estimated for excitation at 695 or 780 nm as appropriate.</p>", "links"=>[], "tags"=>["fluorescence", "properties", "nir"], "article_id"=>318744, "categories"=>["Physiology", "Biotechnology", "Cell Biology", "Physics", "Biophysics"], "users"=>["Christopher J. Tynan", "David T. Clarke", "Benjamin C. Coles", "Daniel J. Rolfe", "Marisa L. Martin-Fernandez", "Stephen E. D. Webb"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036265.t001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ensemble_fluorescence_properties_of_NIR_dyes_/318744", "title"=>"Ensemble fluorescence properties of NIR dyes.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-04-25 02:25:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/334358", "https://ndownloader.figshare.com/files/334389", "https://ndownloader.figshare.com/files/334436", "https://ndownloader.figshare.com/files/334487"], "description"=>"<div><h3>Background</h3><p>The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.</p> <h3>Methodology/Principal Findings</h3><p>A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.</p> <h3>Conclusions/Significance</h3><p>We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.</p> </div>", "links"=>[], "tags"=>["multicolour", "imaging", "cells", "infra-red", "dyes"], "article_id"=>126045, "categories"=>["Physiology", "Biotechnology", "Cell Biology", "Physics", "Biophysics"], "users"=>["Christopher J. Tynan", "David T. Clarke", "Benjamin C. Coles", "Daniel J. Rolfe", "Marisa L. Martin-Fernandez", "Stephen E. D. Webb"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036265.s001", "https://dx.doi.org/10.1371/journal.pone.0036265.s002", "https://dx.doi.org/10.1371/journal.pone.0036265.s003", "https://dx.doi.org/10.1371/journal.pone.0036265.s004"], "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Multicolour_Single_Molecule_Imaging_in_Cells_with_Near_Infra_Red_Dyes/126045", "title"=>"Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-25 01:40:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/648286"], "description"=>"<p>The measured single molecule fluorescence properties of the two brightest NIR dyes tested, in each wavelength band. The mean number of photons per single molecule was calculated from the mean single molecule fluorescence intensity integrated over 250 ms. The laser power exiting the objective was ∼1.6 µWµm<sup>−2</sup> for all dyes. The mean number of photons per single molecule, the mean period of continuous fluorescence emission and the mean duty cycle were obtained from the intensity traces of 74 Alexa 700, 276 IRDye 700DX, 99 Alexa 790 and 272 IRDye 800CW molecules.</p>", "links"=>[], "tags"=>["fluorescence", "properties", "nir"], "article_id"=>318776, "categories"=>["Physiology", "Biotechnology", "Cell Biology", "Physics", "Biophysics"], "users"=>["Christopher J. Tynan", "David T. Clarke", "Benjamin C. Coles", "Daniel J. Rolfe", "Marisa L. Martin-Fernandez", "Stephen E. D. Webb"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036265.t002", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Single_molecule_fluorescence_properties_of_NIR_dyes_/318776", "title"=>"Single molecule fluorescence properties of NIR dyes.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-04-25 02:26:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/648129"], "description"=>"<p><b>A</b>. Images of a single area of unlabelled MCF-7 cells independently illuminated with broadband 780 nm, 695 nm and 545 nm light demonstrate a decrease in autofluorescence background with increasing excitation wavelength. A whitelight transmission image indicates the position of cells within the field of view. <b>B</b>. Images of single molecules of transferrin-IRDye 700DX and transferrin-IRDye 800CW on MCF-7 cells simultaneously illuminated with broadband 780 nm and 695 nm light. Typical intensity vs. time traces, of the molecules highlighted by red and blue circles, are shown to the right. Scale bars represent 2 µm.</p>", "links"=>[], "tags"=>["nir", "molecules", "mcf-7"], "article_id"=>318615, "categories"=>["Physiology", "Biotechnology", "Cell Biology", "Physics", "Biophysics"], "users"=>["Christopher J. Tynan", "David T. Clarke", "Benjamin C. Coles", "Daniel J. Rolfe", "Marisa L. Martin-Fernandez", "Stephen E. D. Webb"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036265.g001", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Autofluorescence_background_and_single_NIR_molecules_in_MCF_7_cells_/318615", "title"=>"Autofluorescence background and single NIR molecules in MCF-7 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:23:35"}

PMC Usage Stats | Further Information

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Relative Metric

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