scholarly journals Generative adversarial network enables rapid and robust fluorescence lifetime image analysis in live cells

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Yuan-I Chen ◽  
Yin-Jui Chang ◽  
Shih-Chu Liao ◽  
Trung Duc Nguyen ◽  
Jianchen Yang ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
High Speed ◽  
Resonance Energy ◽  
Likelihood Estimation ◽  
Live Cells ◽  
Biological Research ◽  
Fluorescence Lifetime Imaging ◽  
Accurate Analysis ◽  
Generative Adversarial Network ◽  
Cellular Environment

AbstractFluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is up to 2,800 times faster than the gold standard time-domain maximum likelihood estimation (TD_MLE) and that flimGANE provides a more accurate analysis of low-photon-count histograms in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis in live cells. With its advantages in speed and reliability, flimGANE is particularly useful in fundamental biological research and clinical applications, where high-speed analysis is critical.

scholarly journals Deep learning enables rapid and robust analysis of fluorescence lifetime imaging in photon-starved conditions

2020 ◽  
Author(s):  
Yuan-I Chen ◽  
Yin-Jui Chang ◽  
Shih-Chu Liao ◽  
Trung Duc Nguyen ◽  
Jianchen Yang ◽  
...  
Keyword(s):  
Deep Learning ◽  
Fluorescence Lifetime ◽  
Resonance Energy ◽  
Excitation Power ◽  
Least Square ◽  
Biological Research ◽  
Fluorescence Lifetime Imaging ◽  
Accurate Analysis ◽  
Lifetime Imaging ◽  
Cellular Environment

AbstractFluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study the molecular states in the complex cellular environment as the lifetime readings are not biased by the fluorophore concentration or the excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is not only 258 times faster than the most popular time-domain least-square estimation (TD_LSE) method but also provide more accurate analysis in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis. With its advantages in speed and reliability, flimGANE is particularly useful in fundamental biological research and clinical applications, where ultrafast analysis is critical.

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 Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

2019 ◽  
Author(s):  
Simone Pelicci ◽  
Alberto Diaspro ◽  
Luca Lanzanò
Keyword(s):  
Dna Damage ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Donor And Acceptor ◽  
Dna Dyes

AbstractChromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

scholarly journals Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

2020 ◽  
Vol 48 (21) ◽  
pp. e122-e122
Author(s):  
Tanja Kaufmann ◽  
Sébastien Herbert ◽  
Benjamin Hackl ◽  
Johanna Maria Besold ◽  
Christopher Schramek ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy ◽  
Population Level ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Uv Laser ◽  
Protein Protein Interactions

Abstract Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.

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

2021 ◽  
Vol 118 (3) ◽  
pp. e2004176118
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

We 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.

Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

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 imaging of transient metabolic dynamics using two-photon fluorescence lifetime imaging microscopy

Optica ◽  
2018 ◽  
Vol 5 (10) ◽  
pp. 1290 ◽  
Author(s):  
Andrew J. Bower ◽  
Joanne Li ◽  
Eric J. Chaney ◽  
Marina Marjanovic ◽  
Darold R. Spillman ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
High Speed ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
High Speed Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence
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