PMID- 34887382 OWN - NLM STAT- MEDLINE DCOM- 20220119 LR - 20230921 IS - 2041-1723 (Electronic) IS - 2041-1723 (Linking) VI - 12 IP - 1 DP - 2021 Dec 9 TI - A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium. PG - 7159 LID - 10.1038/s41467-021-27249-w [doi] LID - 7159 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. CI - (c) 2021. The Author(s). FAU - van der Linden, Franka H AU - van der Linden FH AUID- ORCID: 0000-0001-5324-2714 AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Mahlandt, Eike K AU - Mahlandt EK AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Arts, Janine J G AU - Arts JJG AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. AD - Department of Molecular Hematology at Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. FAU - Beumer, Joep AU - Beumer J AD - Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands. FAU - Puschhof, Jens AU - Puschhof J AD - Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands. FAU - de Man, Saskia M A AU - de Man SMA AUID- ORCID: 0000-0003-0906-5276 AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Chertkova, Anna O AU - Chertkova AO AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Ponsioen, Bas AU - Ponsioen B AD - Center for Molecular Medicine, Oncode Institute, University Medical Centre Utrecht, Utrecht, The Netherlands. FAU - Clevers, Hans AU - Clevers H AUID- ORCID: 0000-0002-3077-5582 AD - Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands. FAU - van Buul, Jaap D AU - van Buul JD AUID- ORCID: 0000-0003-0054-7949 AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. AD - Department of Molecular Hematology at Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. FAU - Postma, Marten AU - Postma M AUID- ORCID: 0000-0002-3167-3633 AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Gadella, Theodorus W J Jr AU - Gadella TWJ Jr AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. FAU - Goedhart, Joachim AU - Goedhart J AUID- ORCID: 0000-0002-0630-3825 AD - Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. j.goedhart@uva.nl. LA - eng PT - Evaluation Study PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20211209 PL - England TA - Nat Commun JT - Nature communications JID - 101528555 RN - 0 (Luminescent Proteins) RN - SY7Q814VUP (Calcium) SB - IM MH - Biosensing Techniques/instrumentation/*methods MH - Calcium/*analysis/metabolism MH - Endothelial Cells/chemistry/*metabolism MH - Fluorescence MH - HeLa Cells MH - Humans MH - Luminescent Proteins/*chemistry/genetics/metabolism MH - Organoids/chemistry/metabolism PMC - PMC8660884 COIS- H.C. is an inventor on patents related to organoid technology. His full disclosure is given at: https://www.uu.nl/staff/JCClevers/Additionalfunctions. The remaining authors declare no competing interests. EDAT- 2021/12/11 06:00 MHDA- 2022/01/20 06:00 PMCR- 2021/12/09 CRDT- 2021/12/10 05:59 PHST- 2021/06/16 00:00 [received] PHST- 2021/11/10 00:00 [accepted] PHST- 2021/12/10 05:59 [entrez] PHST- 2021/12/11 06:00 [pubmed] PHST- 2022/01/20 06:00 [medline] PHST- 2021/12/09 00:00 [pmc-release] AID - 10.1038/s41467-021-27249-w [pii] AID - 27249 [pii] AID - 10.1038/s41467-021-27249-w [doi] PST - epublish SO - Nat Commun. 2021 Dec 9;12(1):7159. doi: 10.1038/s41467-021-27249-w.