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 - Funding Information: F.H.L. was supported by a NWO Chemical Sciences ECHO grant (711.017.003). E.M. was supported by a NWO ALW-OPEN grant (ALWOP.306). M.P. was supported by a NWO-TTP grant (14691). J.v.B. was supported by a ZonMW NOW Vici grant (91819632). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2021, The Author(s).
PY - 2021/12/1
Y1 - 2021/12/1
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.
KW - Biosensing Techniques/instrumentation
KW - Calcium/analysis
KW - Endothelial Cells/chemistry
KW - Fluorescence
KW - HeLa Cells
KW - Humans
KW - Luminescent Proteins/chemistry
KW - Organoids/chemistry
UR - http://www.scopus.com/inward/record.url?scp=85121004330&partnerID=8YFLogxK
UR - https://pure.uva.nl/ws/files/68678274/Supporting_info_41467_2021_27249_MOESM4_ESM.zip
UR - https://pure.uva.nl/ws/files/68678902/41467_2021_27249_MOESM10_ESM.zip
U2 - https://doi.org/10.1038/s41467-021-27249-w
DO - https://doi.org/10.1038/s41467-021-27249-w
M3 - Article
C2 - 34887382
SN - 2041-1723
VL - 12
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 7159
ER -