Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a meth...

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Vydáno v:Nature communications Ročník 13; číslo 1; s. 1850 - 13
Hlavní autoři: Bierbuesse, Franziska, Bourges, Anaïs C., Gielen, Vincent, Mönkemöller, Viola, Vandenberg, Wim, Shen, Yi, Hofkens, Johan, Vanden Berghe, Pieter, Campbell, Robert E., Moeyaert, Benjamien, Dedecker, Peter
Médium: Journal Article
Jazyk:angličtina
Vydáno: London Nature Publishing Group UK 06.04.2022
Nature Publishing Group
Nature Portfolio
Témata:
631/1647/1888/2249
631/1647/245/2225
631/1647/328/2235
639/624/1107/328/2236
639/624/1107/510
Biosensing Techniques - methods
Biosensors
Calcium ions
Dynamical systems
Enzymatic activity
Fluorescence
Fluorescence Resonance Energy Transfer
Fluorescent Dyes
Humanities and Social Sciences
Ionophores
Light
multidisciplinary
Photochromism
Proteins
Science
Science (multidisciplinary)
ISSN:2041-1723, 2041-1723
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Shrnutí:Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca 2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca 2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems. Biosensors often report relative rather than absolute values. Here the authors report a method that utilises the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity: photochromism-enabled absolute quantification (PEAQ) biosensing.
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ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-022-29508-w