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Temporal Filtering to Improve Single Molecule Identification in High Background Samples

Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings...

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Detalles Bibliográficos
Autores principales: Reismann, Alexander W. A. F., Atanasova, Lea, Schrangl, Lukas, Zeilinger, Susanne, Schütz, Gerhard J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321103/
https://www.ncbi.nlm.nih.gov/pubmed/30562966
http://dx.doi.org/10.3390/molecules23123338
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author Reismann, Alexander W. A. F.
Atanasova, Lea
Schrangl, Lukas
Zeilinger, Susanne
Schütz, Gerhard J.
author_facet Reismann, Alexander W. A. F.
Atanasova, Lea
Schrangl, Lukas
Zeilinger, Susanne
Schütz, Gerhard J.
author_sort Reismann, Alexander W. A. F.
collection PubMed
description Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.
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spelling pubmed-63211032019-01-14 Temporal Filtering to Improve Single Molecule Identification in High Background Samples Reismann, Alexander W. A. F. Atanasova, Lea Schrangl, Lukas Zeilinger, Susanne Schütz, Gerhard J. Molecules Article Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable. MDPI 2018-12-17 /pmc/articles/PMC6321103/ /pubmed/30562966 http://dx.doi.org/10.3390/molecules23123338 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Reismann, Alexander W. A. F.
Atanasova, Lea
Schrangl, Lukas
Zeilinger, Susanne
Schütz, Gerhard J.
Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title_full Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title_fullStr Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title_full_unstemmed Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title_short Temporal Filtering to Improve Single Molecule Identification in High Background Samples
title_sort temporal filtering to improve single molecule identification in high background samples
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321103/
https://www.ncbi.nlm.nih.gov/pubmed/30562966
http://dx.doi.org/10.3390/molecules23123338
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