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Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance th...

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Autores principales: Carro-Temboury, Miguel R., Arppe, Riikka, Hempel, Casper, Vosch, Tom, Just Sørensen, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728579/
https://www.ncbi.nlm.nih.gov/pubmed/29236769
http://dx.doi.org/10.1371/journal.pone.0189529
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author Carro-Temboury, Miguel R.
Arppe, Riikka
Hempel, Casper
Vosch, Tom
Just Sørensen, Thomas
author_facet Carro-Temboury, Miguel R.
Arppe, Riikka
Hempel, Casper
Vosch, Tom
Just Sørensen, Thomas
author_sort Carro-Temboury, Miguel R.
collection PubMed
description The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging.
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spelling pubmed-57285792017-12-22 Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission Carro-Temboury, Miguel R. Arppe, Riikka Hempel, Casper Vosch, Tom Just Sørensen, Thomas PLoS One Research Article The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging. Public Library of Science 2017-12-13 /pmc/articles/PMC5728579/ /pubmed/29236769 http://dx.doi.org/10.1371/journal.pone.0189529 Text en © 2017 Carro-Temboury 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
Carro-Temboury, Miguel R.
Arppe, Riikka
Hempel, Casper
Vosch, Tom
Just Sørensen, Thomas
Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title_full Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title_fullStr Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title_full_unstemmed Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title_short Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
title_sort creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728579/
https://www.ncbi.nlm.nih.gov/pubmed/29236769
http://dx.doi.org/10.1371/journal.pone.0189529
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