scholarly journals Selective detection of Cu+ ions in live cells via fluorescence lifetime imaging microscopy

2021 ◽  
Author(s):  
Ramon Vilar ◽  
Martin Priessner ◽  
Peter A. Summers ◽  
Ben Lewis ◽  
Magdalena Sastre ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Selective Detection ◽  
Lifetime Imaging ◽  
Cu Ions

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.

scholarly journals High-speed compressed-sensing fluorescence lifetime imaging microscopy of live cells

2020 ◽  
Author(s):  
Yayao Ma ◽  
Youngjae Lee ◽  
Catherine Best-Popescu ◽  
Liang Gao
Keyword(s):  
Compressed Sensing ◽  
Fluorescence Lifetime ◽  
High Speed ◽  
Time Lapse ◽  
Live Cells ◽  
Large Field ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Specific Domain ◽  
Lifetime Imaging

AbstractWe present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale.

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Peter Andrew Summers ◽  
Ben Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa Maria Porreca ◽  
Aaron H M Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peter A. Summers ◽  
Benjamin W. Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa M. Porreca ◽  
Aaron H. M. Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

AbstractGuanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Sign in / Sign up

Export Citation Format

Share Document