scholarly journals Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 140
Author(s):  
Ting-Yuan Tseng ◽  
Chiung-Lin Wang ◽  
Wei-Chun Huang ◽  
Ta-Chau Chang
Keyword(s):  
Fluorescent Probe ◽  
Fluorescence Lifetime ◽  
Decay Time ◽  
Live Cells ◽  
Great Promise ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Decay Times

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.

scholarly journals Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy

2013 ◽  
Vol 18 (10) ◽  
pp. 101309 ◽  
Author(s):  
Ting-Yuan Tseng ◽  
Cheng-Hao Chien ◽  
Jen-Fei Chu ◽  
Wei-Chun Huang ◽  
Mei-Ying Lin ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Quadruplex Dna ◽  
Lifetime Imaging ◽  
Guanine Quadruplex

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.

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