A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium
| Autor: | Joachim Goedhart, Jens Puschhof, de Man Sm, Janine J. G. Arts, Anna O. Chertkova, Eike K. Mahlandt, Marten Postma, van Buul Jd, van der Linden Fh, Bas Ponsioen, T. W. J. Gadella, Joep Beumer, Hans Clevers |
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| Přispěvatelé: | Hubrecht Institute for Developmental Biology and Stem Cell Research, Landsteiner Laboratory, ACS - Microcirculation, Molecular Cytology (SILS, FNWI), SILS Other Research (FNWI) |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Conformational change Quantitative imaging Science chemistry.chemical_element Quantum yield General Physics and Astronomy Biosensing Techniques Calcium Cellular imaging Calcium in biology General Biochemistry Genetics and Molecular Biology Article Fluorescence imaging Fluorescence Ca2+ imaging Humans Multidisciplinary Chemistry Endothelial Cells General Chemistry Fluorescent proteins Organoids Luminescent Proteins Biophysics Biosensor HeLa Cells |
| Zdroj: | Nature Communications, 12(1). Nature Publishing Group Nature communications, 12(1):7159. Nature Publishing Group Nature Communications Nature Communications, 12:7159. Nature Publishing Group Nature Communications, Vol 12, Iss 1, Pp 1-13 (2021) |
| ISSN: | 2041-1723 |
| Popis: | 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. Currently, genetically encoded calcium indicators are not suitable for direct quantification. Here the authors engineer a fluorescence lifetime imaging calcium biosensor, Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS), and measure intracellular calcium concentrations in human-derived organoids. |
| Databáze: | OpenAIRE |
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