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Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy

Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset...

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Detalles Bibliográficos
Autores principales: Sandoval, Ruben M., Wang, Exing, Molitoris, Bruce A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194064/
https://www.ncbi.nlm.nih.gov/pubmed/25313346
http://dx.doi.org/10.4161/intv.23674
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author Sandoval, Ruben M.
Wang, Exing
Molitoris, Bruce A.
author_facet Sandoval, Ruben M.
Wang, Exing
Molitoris, Bruce A.
author_sort Sandoval, Ruben M.
collection PubMed
description Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.
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spelling pubmed-41940642014-10-11 Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy Sandoval, Ruben M. Wang, Exing Molitoris, Bruce A. Intravital Methods Article Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required. Taylor & Francis 2014-10-20 /pmc/articles/PMC4194064/ /pubmed/25313346 http://dx.doi.org/10.4161/intv.23674 Text en Copyright © 2013 Landes Bioscience https://creativecommons.org/licenses/by-nc/3.0/This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
spellingShingle Methods Article
Sandoval, Ruben M.
Wang, Exing
Molitoris, Bruce A.
Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title_full Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title_fullStr Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title_full_unstemmed Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title_short Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
title_sort finding the bottom and using it: offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
topic Methods Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194064/
https://www.ncbi.nlm.nih.gov/pubmed/25313346
http://dx.doi.org/10.4161/intv.23674
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