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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Franka H. van der Linden ◽  
Eike K. Mahlandt ◽  
Janine J. G. Arts ◽  
Joep Beumer ◽  
Jens Puschhof ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Intracellular Calcium ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Quantitative Imaging ◽  
Fluorescence Lifetime Imaging ◽  
Calcium Dependent ◽  
Dependent Change ◽  
Engineering Strategy ◽  
Robust Measurements

AbstractThe 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.

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

2021 ◽  
Author(s):  
Franka H. van der Linden ◽  
Eike K. Mahlandt ◽  
Janine J.G. Arts ◽  
Joep Beumer ◽  
Jens Puschhof ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Intracellular Calcium ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Quantitative Imaging ◽  
Fluorescence Lifetime Imaging ◽  
Calcium Dependent ◽  
Dependent Change ◽  
Engineering Strategy ◽  
Robust Measurements

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric 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 novel 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. A new 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. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids.

scholarly journals Fluorescence lifetime imaging of pH along the secretory pathway

2021 ◽  
Author(s):  
Peter Linders ◽  
Martin ter Beest ◽  
Geert van den Bogaart
Keyword(s):  
Temporal Resolution ◽  
Fluorescence Lifetime ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Excitation Wavelength ◽  
Fluorescence Lifetime Imaging ◽  
Ph Homeostasis ◽  
Ph Changes ◽  
Lifetime Imaging ◽  
Wide Range

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway. Then, we analyze the dynamic pH changes within cells treated with Brefeldin A, a COPI coat inhibitor. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)-α while it was in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

Fluorescence lifetime imaging of intracellular calcium

1993 ◽  
Vol 3 (3) ◽  
pp. 161-167 ◽  
Author(s):  
Henryk Szmacinski ◽  
Joseph R. Lakowicz ◽  
W. J. Lederer ◽  
K. Nowaczyk ◽  
Michael L. Johnson
Keyword(s):  
Intracellular Calcium ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging

2013 ◽  
Vol 405 (12) ◽  
pp. 3983-3987 ◽  
Author(s):  
Sandrine Poëa-Guyon ◽  
Hélène Pasquier ◽  
Fabienne Mérola ◽  
Nicolas Morel ◽  
Marie Erard
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Protein A ◽  
Ph Sensor ◽  
Cyan Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Enhanced Cyan Fluorescent Protein

scholarly journals An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17896 ◽  
Author(s):  
Michele L. Markwardt ◽  
Gert-Jan Kremers ◽  
Catherine A. Kraft ◽  
Krishanu Ray ◽  
Paula J. C. Cranfill ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Site Directed Mutagenesis ◽  
Quantum Yields

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

scholarly journals Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

2012 ◽  
Vol 48 (42) ◽  
pp. 5127 ◽  
Author(s):  
Antonella Battisti ◽  
Michelle A. Digman ◽  
Enrico Gratton ◽  
Barbara Storti ◽  
Fabio Beltram ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

scholarly journals Unravelling the mechanisms controlling heme supply and demand

2021 ◽  
Vol 118 (22) ◽  
pp. e2104008118
Author(s):  
Galvin C.-H. Leung ◽  
Simon S.-P. Fung ◽  
Andrea E. Gallio ◽  
Robert Blore ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Supply And Demand ◽  
Fluorescence Lifetime Imaging ◽  
Prosthetic Group ◽  
Lifetime Imaging ◽  
Free Heme ◽  
Green Fluorescent ◽  
Cellular Compartments

In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

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