Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Oct 8;10:16843. doi: 10.1038/s41598-020-73987-0

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Determining the range of glutathione redox potential E_GSH values we can measure accurately with the roGFP1-R12 biosensor. (a) Glutathione redox potential (E_GSH) directs the oxidation of cysteines in hundreds of proteins in the same direction, resulting in their concerted regulation. (b) The reduced and oxidized states of the roGFP1-R12 biosensor have different fluorescence spectra⁸, enabling E_GSH measurement via R (fluorescence ratio) microscopy. (c) The conversion map from R to E_GSH is highly nonlinear. R_{reduced state} and R_{oxidized state} refer to the ratiometric emission of ensembles of reduced and oxidized biosensors, respectively. E⁰^′ is the standard midpoint potential of the biosensor. (d) The top panel shows how measurement errors in R cause observed E_GSH values (E_Obs) to differ from the true E_GSH values (E_True) that would be observed if R was measured with no error (R_True). The bottom panel shows how the size of an E_GSH error (E_Obs – E_True) depends not only on the size of the error in R but also on the value of R. Each dotted curve corresponds to a different fold-change error in R. The shaded region corresponds the interval encompassing 95% of the predicted E_Obs values for each R_True value, given our empirical error in R. (e) Transforming the map from R_True to E_True in the top and bottom panels shown in (d) produces plots showing how errors in R influence the map from E_True to E_Obs (top panel) and how the size of an E_GSH error depends not only on the size of the error in R but also on the value of E_True (bottom panel). Each dotted curve corresponds to a different fold-change error in R. The shaded region shows the interval encompassing 95% of the predicted E_Obs values for each E_True value, given our empirical error in R. (f) Cumulative distribution of the empirical fold error in R in live C. elegans expressing the roGFP1-R12 biosensor in the cytosol of the anterior (pm3) muscles of the pharynx, the feeding organ. This error distribution was obtained by aggregating with equal weight the empirical fold error in R of five separate experiments (see Supplementary Note 3). 95% of the errors in R fall within the interval (− 2.8%, + 2.8%), shown shaded in gray. This interval quantifies the precision of our fluorescence-ratio measurements. (g) E_GSH measurement inaccuracy (the maximum absolute difference between E_True and E_Obs) decreases with increased precision of R measurement. Each dotted curve corresponds to a different precision of R measurement. The shaded region shows the interval encompassing 95% of the predicted E_GSH measurement inaccuracies for each E_True value, given our empirical error in R.