Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry
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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2077215"], "description"=>"<p>(a) Three state model with rates. (b) Cutout of total image of sparsely distributed DNA oligomers on glass labeled with single Alexa Fluor 647 dyes showing well-isolated clusters of localizations. (c) Cumulative number of localizations and single-exponential fit. (d) Correlation parameter <i>Q</i> determined from the spatial image correlations and fit with switching model shows agreement with the ground truth value determined from the cluster analysis. The estimated value for the average number of localizations ⟨<i>M</i>(<i>t</i>)⟩ shows agreement with the ground truth value determined from the cluster statistics. (e-g) Histograms of the number of localizations accumulated per cluster and model prediction at three time points during the image acquisition.</p>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421824, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0127989.g001", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_localization_microscopy_with_a_single_fluorophore_per_labeled_site_/1421824", "title"=>"Quantitative localization microscopy with a single fluorophore per labeled site.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-20 04:42:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/2077248", "https://ndownloader.figshare.com/files/2077249", "https://ndownloader.figshare.com/files/2077250", "https://ndownloader.figshare.com/files/2077251", "https://ndownloader.figshare.com/files/2077252", "https://ndownloader.figshare.com/files/2077253", "https://ndownloader.figshare.com/files/2077254"], "description"=>"<div><p>Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.</p></div>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421857, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0127989.s001", "https://dx.doi.org/10.1371/journal.pone.0127989.s002", "https://dx.doi.org/10.1371/journal.pone.0127989.s003", "https://dx.doi.org/10.1371/journal.pone.0127989.s004", "https://dx.doi.org/10.1371/journal.pone.0127989.s005", "https://dx.doi.org/10.1371/journal.pone.0127989.s006", "https://dx.doi.org/10.1371/journal.pone.0127989.s007"], "stats"=>{"downloads"=>7, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_Localization_Microscopy_Effects_of_Photophysics_and_Labeling_Stoichiometry_/1421857", "title"=>"Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-05-20 04:42:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/2077222"], "description"=>"<p>(a) FCS-analysis of NB stoichiometry indicating there is a single fluorophore per NB. (b) Cutout of quantitative localization microscopy image of NB-labeled Seh1 in the NPC (<i>k</i><sub><i>bl</i></sub> = 4.8 × 10<sup>−3</sup>/s and <i>M</i><sub>∞</sub> = 5.0). The numbers at the green boxes indicate the estimated number of NBs within the box. (c) Histogram of the estimated number of NBs per NPC.</p>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421831, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0127989.g002", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_localization_microscopy_of_NB_labeled_Seh1_in_the_NPC_/1421831", "title"=>"Quantitative localization microscopy of NB-labeled Seh1 in the NPC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-20 04:42:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/2077224"], "description"=>"<p>(a) Number of localizations per neutravidin tetramer as a function of DOL as estimated from the image correlations and the ground truth values from cluster analysis, showing good agreement. (b) Fitted bleach rate <i>k</i><sub><i>bl</i></sub> and switching rate <i>k</i><sub><i>sw</i></sub> = <i>M</i><sub>∞</sub><i>k</i><sub><i>bl</i></sub> as a function of DOL values for the same data, indicating independent activation and bleaching per label. Error bars indicate the standard deviation among samples at the same DOL. (c) Image of IgE receptors on the membrane of RBL cells labeled with primary antibodies with a DOL of 1.5 (<i>k</i><sub><i>bl</i></sub> = 9.1 × 10<sup>−3</sup>/s and <i>M</i><sub>∞</sub> = 2.3).</p>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421833, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0127989.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_localization_microscopy_with_multiple_emitters_per_labeled_site_/1421833", "title"=>"Quantitative localization microscopy with multiple emitters per labeled site.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-20 04:42:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/2077228"], "description"=>"<p>(a) Overview image (pixel size 10 nm, clipped for visibility) and (b) zoomed inset (pixel size 4 nm) of the dashed white box in (a) of secondary antibody-Alexa Fluor 647 labeled Nup153 protein of the NPC in the nuclear membrane with non-specifically bound (secondary) antibodies outside the nuclear membrane region. (c) The correlation parameter Q for the region inside the nuclear membrane (red box) is higher than outside (blue box) due to the tight clustering of the secondary antibodies labeling the Nup153 proteins. The relative number of accumulated localizations at each time point is similar, indicating that the bleaching behavior is similar and the sources of the localizations are identical in both regions.</p>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421837, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0127989.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_localization_microscopy_with_heterogeneous_labeling_density_/1421837", "title"=>"Quantitative localization microscopy with heterogeneous labeling density.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-20 04:42:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/2077229"], "description"=>"<p>Localizations were filtered for the minimum number of photons per event before grouping, minimum number of photons per event after grouping, the maximum duration of the event after grouping, and the maximum width (FWHM) of the Gaussian fitted to the spot.</p>", "links"=>[], "tags"=>["localization microscopy", "transition rates", "quantification method", "Stoichiometry Quantification", "reversibly switchable fluorophores", "stoichiometry", "DNA oligomers", "Quantitative Localization Microscopy", "image correlation"], "article_id"=>1421838, "categories"=>["Uncategorised"], "users"=>["Robert P. J. Nieuwenhuizen", "Mark Bates", "Anna Szymborska", "Keith A. Lidke", "Bernd Rieger", "Sjoerd Stallinga"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0127989.t001", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Parameters_used_for_filtering_localization_events_/1421838", "title"=>"Parameters used for filtering localization events.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-05-20 04:42:31"}

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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