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

2019 ◽  
Author(s):  
Nur P. Damayanti ◽  
Sampreeti Jena ◽  
Jackie Tan ◽  
Joseph M. K. Irudayaraj ◽  
L. L. Parker
Keyword(s):  
Protein Expression ◽  
Fluorescence Lifetime ◽  
Kinase Activity ◽  
Src Family Kinases ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Peptide Substrates ◽  
Lifetime Imaging ◽  
Cell Penetrating ◽  
Preliminary Application

Most commonly employed strategies to map kinase activities in live cells require expression of genetically-encoded proteins (e.g. FRET sensors). In this work, we describe development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We also demonstrate image-by-image and pixel-by-pixel quantification of probe phosphorylation ratio, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular kinase activity in live cells.

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 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.

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 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
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