scholarly journals Fluorescence lifetime imaging of compartmental pH dynamics using red fluorescent protein sensors in live cells

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Emily Haynes ◽  
Megha Rajendran ◽  
Angeline Lyon ◽  
Nicholas Noinaj ◽  
Richard Day ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Red Fluorescent Protein ◽  
Lifetime Imaging ◽  
Protein Sensors

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.

scholarly journals Streamlined histone-based fluorescence lifetime imaging microscopy reveals ATM regulation of chromatin compaction

2017 ◽  
Author(s):  
Alice Sherrard ◽  
Paul Bishop ◽  
Melanie Panagi ◽  
Maria Beatriz Villagomez ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fluorescence Lifetime ◽  
Genotoxic Stress ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
Lifetime Imaging ◽  
Detailed Method ◽  
Chromatin Organisation

AbstractChanges in chromatin compaction are crucial during genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome function. Here, we address this challenge by developing, validating, and streamlining histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells; in 2D and 3D. We present quality-controlled and detailed method that is simpler and faster than previous approches, and uses FLIMfit open-source software. We demonstrate the versatility of our method through its combination with immunofluorescence and its implementation in immortalised cells and primary neurons. Owing to these developments, we applied this method to elucidate the function of the DNA damage response kinase, ATM, in regulating chromatin organisation after genotoxic-stress. We unravelled a role for ATM in regulating chromatin compaction independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure in cells, and establish its broader utility.

Fluorescence lifetime imaging of microviscosity changes during ER autophagy in live cells

2018 ◽  
Author(s):  
Ying He ◽  
Junle Qu ◽  
Soham Samanta ◽  
Wufan Liu ◽  
Wenhui Pan ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

Spectroscopy and fluorescence lifetime imaging in live cells of a cyano-substituted combretastatin

2014 ◽  
Vol 3 (3) ◽  
pp. 211-218 ◽  
Author(s):  
Kathrin M. Scherer ◽  
Roger H. Bisby ◽  
Stanley W. Botchway ◽  
Greg M. Greetham ◽  
John A. Hadfield ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

scholarly journals Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

2013 ◽  
Vol 52 (14) ◽  
pp. 3931-3934 ◽  
Author(s):  
Nur P. Damayanti ◽  
Laurie L. Parker ◽  
Joseph M. K. Irudayaraj
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Peptide Phosphorylation

scholarly journals Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases

2020 ◽  
Vol 56 (87) ◽  
pp. 13409-13412
Author(s):  
Sampreeti Jena ◽  
Nur P. Damayanti ◽  
Jackie Tan ◽  
Erica D. Pratt ◽  
Joseph M. K. Irudayaraj ◽  
...  
Keyword(s):  
Single Cell ◽  
Fluorescence Lifetime ◽  
Src Family Kinases ◽  
Live Cells ◽  
Cell Penetrating Peptide ◽  
Fluorescence Lifetime Imaging ◽  
Src Family Kinase ◽  
Peptide Substrates ◽  
Lifetime Imaging ◽  
Cell Penetrating

Here we describe development of cell-penetrating peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities, applied to mapping single cell and subcellular dynamics of multiple kinases in live cells.

scholarly journals Fluorescence lifetime imaging of intracellular magnesium content in live cells

The Analyst ◽  
2019 ◽  
Vol 144 (6) ◽  
pp. 1876-1880 ◽  
Author(s):  
Azzurra Sargenti ◽  
Alessia Candeo ◽  
Giovanna Farruggia ◽  
Cosimo D'Andrea ◽  
Concettina Cappadone ◽  
...  
Keyword(s):  
Detailed Analysis ◽  
Fluorescence Lifetime ◽  
Cell Imaging ◽  
Magnesium Content ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Intracellular Magnesium ◽  
Lifetime Imaging

The first detailed analysis for Mg cell imaging by visible FLIM is presented.

scholarly journals Fluorescence lifetime imaging of physiological free Cu(ii) levels in live cells with a Cu(ii)-selective carbonic anhydrase-based biosensor

Metallomics ◽  
2014 ◽  
Vol 6 (5) ◽  
pp. 1034 ◽  
Author(s):  
Bryan J. McCranor ◽  
Henryk Szmacinski ◽  
Hui Hui Zeng ◽  
Andrea K. Stoddard ◽  
Tamiika Hurst ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Fluorescence Lifetime ◽  
Live Cells ◽  
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
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