TY - JOUR
T1 - A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium
AU - van der Linden, Franka H.
AU - Mahlandt, Eike K.
AU - Arts, Janine J.G.
AU - Beumer, Joep
AU - Puschhof, Jens
AU - de Man, Saskia M.A.
AU - Chertkova, Anna O.
AU - Ponsioen, Bas
AU - Clevers, Hans
AU - van Buul, Jaap D.
AU - Postma, Marten
AU - Gadella, Theodorus W.J.
AU - Goedhart, Joachim
N1 - Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85121004330&partnerID=8YFLogxK
U2 - 10.1038/s41467-021-27249-w
DO - 10.1038/s41467-021-27249-w
M3 - Article
C2 - 34887382
AN - SCOPUS:85121004330
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 7159
ER -