A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

Franka H van der Linden, Eike K Mahlandt, Janine J G Arts, Joep Beumer, Jens Puschhof, Saskia M A de Man, Anna O Chertkova, Bas Ponsioen, Hans Clevers, Jaap D van Buul, Marten Postma, Theodorus W J Gadella, Joachim Goedhart

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgaveArtikelWetenschappelijkpeer review

Samenvatting

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

Originele taal-2Engels
Pagina's (van-tot)7159
TijdschriftNature Communications
Volume12
Nummer van het tijdschrift1
DOI's
StatusGepubliceerd - 09 dec. 2021

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