Fluorescence Lifetime of Fluorescent Proteins

2011 ◽  
pp. 69-97 ◽  
Author(s):  
Gregor Jung ◽  
Andreas Brockhinke ◽  
Thomas Gensch ◽  
Benjamin Hötzer ◽  
Stefanie Schwedler ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Proteins

scholarly journals Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins

PLoS ONE ◽  
2018 ◽  
Vol 13 (11) ◽  
pp. e0208075 ◽  
Author(s):  
Laura Canty ◽  
Santosh Hariharan ◽  
Qian Liu ◽  
Steven A. Haney ◽  
David W. Andrews
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Proteins ◽  
Emission Wavelength ◽  
Peak Emission Wavelength ◽  
Peak Emission

Fluorescence Lifetime of Fluorescent Proteins as an Intracellular Environment Probe Sensing the Cell Cycle Progression

2012 ◽  
Vol 7 (8) ◽  
pp. 1385-1392 ◽  
Author(s):  
Artem Pliss ◽  
Lingling Zhao ◽  
Tymish Y. Ohulchanskyy ◽  
Junle Qu ◽  
Paras N. Prasad
Keyword(s):  
Cell Cycle ◽  
Fluorescence Lifetime ◽  
Cell Cycle Progression ◽  
Fluorescent Proteins ◽  
Cycle Progression ◽  
Intracellular Environment

scholarly journals Red-Shifted Fluorescent Proteins Improve FRET Biosensors for High-throughput Fluorescence Lifetime Screening

2018 ◽  
Vol 114 (3) ◽  
pp. 171a
Author(s):  
Tory Schaaf ◽  
Ang Li ◽  
Benjamin Grant ◽  
Prachi Bawaskar ◽  
Evan Kleinboehl ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Lifetime ◽  
Fluorescent Proteins ◽  
Fret Biosensors

scholarly journals Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

2007 ◽  
Vol 40 (6) ◽  
pp. 1105-1112 ◽  
Author(s):  
Antoine Royant ◽  
Philippe Carpentier ◽  
Jérémy Ohana ◽  
John McGeehan ◽  
Bernhard Paetzold ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Single Photon ◽  
Fluorescent Proteins ◽  
Fluorescence Emission ◽  
Spectroscopic Techniques ◽  
Fluorescence Emission Spectrum ◽  
X Ray Diffraction ◽  
Lifetime Measurements ◽  
X Ray ◽  
Solution State

Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV–visible absorbance or fluorescence mode have evolved over the past few decades to become the instruments of choice to perform such tests. Described here are recent improvements to the microspectrophotometer of the so-called Cryobench laboratory located at the European Synchrotron Radiation Facility, Grenoble, France. Optical and mechanical properties have been enhanced so as to record better spectra on smaller samples. A device has been implemented to measure the signal decay of fluorescent samples, either in the crystalline or in the solution state. Recording of the fluorescence lifetime in addition to the steady-state fluorescence emission spectrum allows precise monitoring of the fluorescent sample under study. The device consists of an adaptation of a commercially available time-correlated single-photon-counting (TCSPC) system. A method to record and analyze series of TCSPC histograms,e.g.collected as a function of temperature, is described. To validate the instruments, fluorescence lifetimes of fluorescent small molecules or proteins in the crystalline or solution state, at room and cryo temperatures, have been measured. Lifetimes of a number of fluorescent proteins of the GFP family were generally found to be shorter in crystals than in solution, and slightly longer at cryo temperatures than at ambient temperature. The possibility of performing fluorescence lifetime measurements on crystals at synchrotron facilities widens the variety of spectroscopic techniques complementing X-ray diffraction on macromolecular crystallography beamlines.

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.

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