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PLATERO: A calibration protocol for plate reader green fluorescence measurements

One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by a certain fluorescent molecule, such as the Green Fluorescent Protein (GFP). However, these measurements are...

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Autores principales: González-Cebrián, Alba, Borràs-Ferrís, Joan, Boada, Yadira, Vignoni, Alejandro, Ferrer, Alberto, Picó, Jesús
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9895789/
https://www.ncbi.nlm.nih.gov/pubmed/36741754
http://dx.doi.org/10.3389/fbioe.2023.1104445
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author González-Cebrián, Alba
Borràs-Ferrís, Joan
Boada, Yadira
Vignoni, Alejandro
Ferrer, Alberto
Picó, Jesús
author_facet González-Cebrián, Alba
Borràs-Ferrís, Joan
Boada, Yadira
Vignoni, Alejandro
Ferrer, Alberto
Picó, Jesús
author_sort González-Cebrián, Alba
collection PubMed
description One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by a certain fluorescent molecule, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we describe PLATERO, a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression that returns normalized, gain-independent, and comparable data, even if the acquisition was done at different device gain levels. Most important, the PLATERO embeds a Linearity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluates the sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, and perform the uncertainty and variability assessment are available in an open access repository.
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spelling pubmed-98957892023-02-04 PLATERO: A calibration protocol for plate reader green fluorescence measurements González-Cebrián, Alba Borràs-Ferrís, Joan Boada, Yadira Vignoni, Alejandro Ferrer, Alberto Picó, Jesús Front Bioeng Biotechnol Bioengineering and Biotechnology One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by a certain fluorescent molecule, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we describe PLATERO, a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression that returns normalized, gain-independent, and comparable data, even if the acquisition was done at different device gain levels. Most important, the PLATERO embeds a Linearity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluates the sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, and perform the uncertainty and variability assessment are available in an open access repository. Frontiers Media S.A. 2023-01-20 /pmc/articles/PMC9895789/ /pubmed/36741754 http://dx.doi.org/10.3389/fbioe.2023.1104445 Text en Copyright © 2023 González-Cebrián, Borràs-Ferrís, Boada, Vignoni, Ferrer and Picó. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
González-Cebrián, Alba
Borràs-Ferrís, Joan
Boada, Yadira
Vignoni, Alejandro
Ferrer, Alberto
Picó, Jesús
PLATERO: A calibration protocol for plate reader green fluorescence measurements
title PLATERO: A calibration protocol for plate reader green fluorescence measurements
title_full PLATERO: A calibration protocol for plate reader green fluorescence measurements
title_fullStr PLATERO: A calibration protocol for plate reader green fluorescence measurements
title_full_unstemmed PLATERO: A calibration protocol for plate reader green fluorescence measurements
title_short PLATERO: A calibration protocol for plate reader green fluorescence measurements
title_sort platero: a calibration protocol for plate reader green fluorescence measurements
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9895789/
https://www.ncbi.nlm.nih.gov/pubmed/36741754
http://dx.doi.org/10.3389/fbioe.2023.1104445
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