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20 results for “fluorescence lifetime”
Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy
<p>FRET-based methods are a special tool for detecting interactions between (bio)molecules and their immediate environment. The spatial distribution of molecular interactions and functional states can be seen using FLIM (Fluorescence Lifetime IMaging) and FRET imaging. The spatial information, accuracy, and dynamic range of the observed signals are, however, constrained by the fact that conventional FLIM and FRET imaging only provides average information over an ensemble of molecules within a diffraction-limited volume. On the other hand, conventional Single Molecule Localization Microscopy (SMLM) relies on highly sensitive multi-pixel detectors (e.g. sCMOS or EM-CCD) whose time resolution is not suitable for fluorescence lifetime measurements.</p> <p>Here, we demonstrate a method for obtaining super-resolved FRET imaging using confocal fluorescence-lifetime single-molecule localization microscopy. The proof of concept was carried out using a DNA origami sample for performing DNA-PAINT measurements in combination with fluorogenic probes for reducing background signal. With this method, We show that FRET events separated by sub-diffraction distances can be distinguished based on lifetime modifications.</p>
Raw data supporting "mScarlet fluorescence lifetime reports lysosomal pH quantitatively"
<p>Original dataset and processing code supporting the preprint (scientific publication) "mScarlet fluorescence lifetime reports lysosomal pH quantitatively."</p> <p>Publication Abstract: The lysosome maintains a highly acidic pH, which is critical for successful lysosomal catabolism. Lysosomal pH (pHlys) is difficult to measure because of the simultaneous need for a sensor with large dynamic range, genetic targetability, low pKa, and a quantitative readout. Here, we demonstrate that the fluorescence lifetime of the mScarlet-LAMP1 fusion protein quantitatively reports lysosomal pH, exhibiting a large dynamic range and a pKa well-tuned for the lysosome. Because fluorescence lifetime is an intrinsic property, pH measurements can be achieved in a single fluorescence channel. mScarlet-LAMP1 lifetime allows for individual lysosome-resolved recordings, a critical advance in describing and understanding pHlys heterogeneity. Using this biosensor, we quantify heterogeneity of pHlys in cultured cells at rest and over time during drug treatment. We anticipate that mScarlet-LAMP1 will enable new insights into the diversity of lysosomal physiology and ionic milieu.</p>
Insights into metabolic changes during epidermal differentiation as revealed by multiphoton microscopy with fluorescence lifetime imaging
<p>Rapid developments in the field of organotypic cultures has generated a growing need for effective quality control measures during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied as a non-invasive approach to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. Keratinocytes grown at 1.5 mM Ca2+ exhibited increased expression of differentiation markers KRT1 and KRT10 compared to 60 μM Ca2+, and a metabolic shift from glycolysis to mitochondrial respiration. Fitting the fluorescence decay with a biexponential model revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Using these two parameters, the epidermal differentiation process could be monitored over a 96 h period. Implementing discriminating analysis based on k-means clustering generated clusters that correlated well with culturing time, suggesting that this methodology can be employed as part of an automated pipeline for monitoring keratinocyte differentiation.</p>
SupportingDataset Identification and Quantification of Within-Burst Dynamics in Singly-Labeled Single-Molecule Fluorescence Lifetime Experiments
<p>The Jupyter notebooks and resulting files used to demonstrate divisor-based mpH<sup>2</sup>MM. The analysis is demonstrated with both simulations and analyses of alpha-synuclein.</p> <ol> <li> <p><em><strong>Notebooks.zip</strong></em>:* Zip file containing the Jupyter notebooks for producing, analyzing and visualizing the simulated photon trajectories. Note: this folder contains all code needed to reproduce simulations. All other files related to the simulations are produced by one of the notebooks in this trajectory. However, as simulations can take a long time, the various results files are included in this repository so that notebooks can be run from intermediate steps.</p> <ol> <li> <p><strong>1-PIFE-pybromo-sims.ipynb</strong> : The code for producing simulated diffusion trajectories and photon-HDF5 files of two-state systems undergoing transition dynamics (the results of this notebook are stored in the sub-folder <em>PyBroMo_photonHDF5</em>)</p> </li> <li> <p><strong>2-PIFE-mpH2MM-sim-[lifetime components].ipynb </strong>: Notebooks performing divisor-based mpH<sup>2</sup>MM on simulated datasets for a given combination of lifetime states. (these notebooks store files that are contained in the sub-folder <em>H2MMresults</em>)</p> </li> <li> <p><strong>3-PIFE-mpH2MM-compiled-plots.ipynb</strong> : Jupyter notebook for producing figures comparing all results globally</p> </li> <li> <p><strong>532nm_IRF_19-10-2021.csb</strong>: the file containing the experimental IRF used in the simulations</p> </li> </ol> </li> <li> <p><em><strong>PyBroMo_photonHDF5.zip</strong></em>:* Zip file containing the simulated results of <em>1-PIFE-pybromo-sims</em> notebook as photon-HDF5 files (1 file per transition rate/lifetime combination)</p> </li> <li> <p><strong>PIFE-sim-dynamicmix_[lifetime components]_result.hdf5</strong>: special HDF5 files containing the results of each notebook in <em>Notebooks</em>, which are used by <em>3-PIFE-mpH2MM-compiled-plots</em></p> </li> <li> <p><strong>PIFE-mpH2MM-alpha-syn-vFinal.ipynb</strong>: divisor-based mpH<sup>2</sup>MM analysis of alpha-synuclein smPIFE data</p> </li> <li> <p><strong>H2MM-Lifetime_example.ipynb</strong>: A demonstration of divisor-based mpH<sup>2</sup>MM using nsALEX-smFRET data. This method could potentially demonstrate states differentiated in lifetimes independently of potential changes in E & S.</p> </li> <li> <p><strong>Template_ltH2MM.ipynb</strong>: An easy-to-follow implementation of divisor-based mpH<sup>2</sup>MM demonstrated on a single alpha-synuclein experimental data acquisition file. This can be used for learning how to implement and analyze single dye fluorescence lifetime data with mpH<sup>2</sup>MM</p> </li> </ol> <p> </p> <p>* For running these notebooks, generally, all files in <em>PyBroMo_photonHDF5.zip</em> should be placed into a single directory (i.e., the files in <em>Notebooks</em>.<em>zip</em> should be placed into the same directory as the files in <em>PyBroMo_photonHDF5</em>.<em>zip</em>) as the notebooks are set to read in files from their current directory.</p>
Sparks et al, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy: In vitro data
<p>Data is divided into three folders:</p> <ul> <li>Sparks_et_al_FIG2_Histone_vs_free_GFP <ul> <li>data for Sparks et al Figure 2</li> <li>main text section: <em>'FRET between chromatin-bound GFP and doxorubicin'</em></li> </ul> </li> <li>Sparks_et_al_FIG3_in_vitro_dose_response <ul> <li>data for Sparks et al Figure 3#</li> <li>main text section:<em> 'FLIM endomicroscope can monitor doxorubicin cellular uptake'</em></li> </ul> </li> <li>Sparks_et_al_SuppFIG2_endoscope_spectral_cross_talk <ul> <li>data for Sparks et al Supplementary Figure 2</li> <li>Supplementary information</li> </ul> </li> </ul> <p><strong>Cell lines</strong></p> <p>IGROV-1 cell lines were cultured in CO<sub>2</sub> dependent media with 10% fetal bovine serum and 1% Pen Strep at 37 ˚C. Before experiments, cells were grown to 80% confluence. For measuring doxorubicin uptake by fluorescence an IGROV-1 cell line stably expressing GFP fused to Histone-1 (H1) was made using the PiggyBac transposon system. As a control to show that effect of doxorubicin on GFP depends on whether it is fused to H1 or not, a stable whole cell expression of GFP by lentiviral transfection and selection by Geneticin was made. For bioluminescence imaging of xenograft tumors, all IGROV-1 cell lines were made to stably express firefly luciferase.</p> <p>To investigate the effect of doxorubicin on other histones, IGROV-1 cells were transiently transfected with a Histone-2B-GFP plasmid (gift from Kurt Anderson) using the Lipofectamine® 2000 reagent.</p> <p>IGROV-1 cells were obtained from Crick institute cell services and confirmed as IGROV-1 by Short Tandem Repeats (STR) profiling and no mycoplasma was detected.</p> <p><strong>In vitro experiments</strong></p> <p>IGROV-1 cells were grown to 80% confluence in 75 ml flasks before being re-plated in 12 or 24 well plates or 35 ml glass bottomed dishes and allowed to attach to the surface for 24 hours before experiments.</p> <p>To study how the fluorescence of GFP labelled H1 labelled IGROV-1 cells changes with doxorubicin treatment, fluorescence intensity and lifetime distributions were measured from cells after 3 hours of incubation with doxorubicin of varying concentrations (0, 0.18, 0.9, 1.8, 9, 18 µM) by serial dilutions of a stock solution with PBS. After 3 of hours, cells were washed in PBS then fixed for 20 minutes in 4% PFA. Cells were then imaged in PBS. Doxorubicin hydrochloride (Sigma-Aldrich, D1515-10 mg) was dissolved in PBS to a concentration of 9 mM and stored at -20˚C.</p>
Sparks et al, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy: in vivo data
<p>Data is divided into three folders:</p> <ul> <li>Sparks_et_al_FIG_6_IP_intranodule_heterogeneity <ul> <li>data for Sparks et al Figure 6</li> <li>main text section: <em>'FRET between chromatin-bound GFP and doxorubicin'</em></li> </ul> </li> <li>Sparks_et_al_FIG4_5_6_IP_IV_chemo_comparison <ul> <li>data for Sparks et al Figures 4,5 & 6</li> <li>main text section:<em> 'FLIM endomicroscope can monitor doxorubicin cellular uptake'</em></li> </ul> </li> <li>Sparks_et_al_FIG6_IP__internodule_heterogeneity <ul> <li>data for Sparks et al Figure 6</li> <li>main text section: <em>'Intra-tumor heterogeneity'</em></li> </ul> </li> </ul> <p><strong>In vivo experiments</strong></p> <p>Murine xenografts were prepared by intraperitoneal (IP) injection of IGROV-1 cancer cells. IGROV-1 cells were grown to 80% confluence before being trypsinized and re‑suspended in PBS at a concentration of cells per ml. cells were injected into ICRF nude mice. After 14 days post-injection, the presence of intraperitoneal tumors was confirmed by bioluminescence imaging. Briefly, an IVIS bioluminescence imaging system was used to image isoflurane anesthetized mice. 100 µl of D-luciferin (luciferase substrate) at 30mg ml<sup>-1</sup> was injected IP 10 minutes before recording of bioluminescence images. The presence of peritoneal tumors was confirmed if bioluminescence signals from the peritoneum were above background noise 10-30 minutes after D‑luciferin injections. Following confirmation of tumors, in vivo fluorescence imaging experiments were carried out after 21 days. To study differences in drug uptake between intravenous or intraperitoneal delivery, prior to imaging mice were subject to IP or IV doxorubicin-based chemotherapy for 1.5, 3 or 24 hours. Imaging involved terminal procedures, mice were anesthetized then peritoneal tumors were exposed by minor surgery and inspected with the CEM.</p> <p>All animal model procedures were approved by The Francis Crick Institute Biological Ethics Committee and UK Home Office authority provided by Project License 70/8380.</p> <p> </p> <p> </p>
Example of Fluorescence Lifetime Imaging Microscopy (FLIM) image stack in .ptu format
<p>The dataset is a 3D stack of fluorescence lifetime imaging microscopy (FLIM) images in ptu format to be used as test and training data. It contains the original .lif file (1) with the stack and a single plane image (to be opened using LAS X and LAS X SMD FLIM), exported raw FLIM data in .ptu format of the stack (3) and the single plane (2a) (to be opened in software capable of reading .ptu files) as well as an intensity image in .tif format (2b) of the single plane for a quick sample overview.</p> <p>The sample is a cross-section of hazel (<em>Corylus avellana</em>) 'diclinous male flower t.s.' with Etzold staining provided by the company Zeiss (CZ 01/05). The dataset was generated using a Leica Stellaris 8 upright confocal laser scanning microscope using a 93x/1.4 glycerol immersion objective. Each image of the 65 slice stack with z step size of 0.287 µm contains 512 x 512 pixels with a pixel size of 0.078 µm x 0.078 µm. Excitation was done with a white-light laser at 491 nm and a laser pulse rate of 40 MHz and a pixel dwell time of 2.0875 µs. Images were acquired using a HyD X detector in counting mode in the spectral range of 496 to 739 nm using Leica Application Suite X (LAS X) version 4.4.0.24861 and LAS X SMD FLIM version 4.5.0 for FLIM image acquisition. 10 frames were accumulated per image. Metadata is available as text file (4a) and as metadata files from LAS X (4b).</p>
Dataset related to article "Evaluation of cell metabolic adaptation in wound and tumour by fluorescence Lifetime imaging Microscopy"
<p>This record contains data related to article "Evaluation of cell metabolic adaptation in wound and tumour by fluorescence Lifetime imaging Microscopy"</p> <p>Abstract</p> <p>Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. The pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of NADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer.</p>
Data for "Heat treatment and fiber drawing effect on the matrix structure and fluorescence lifetime of Er- and Tm-doped silica optical fibers"
<p>Includes data for absorption and attenuation measurements and calculations, profiles of refractive index and concentrations, TEM images, XRD patters, and data for fluorescence decay curves presented in the graphs.</p>
Fluorescence lifetime imaging of pH along the secretory pathway
<p>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.</p>
Comprehensive database of fluorescence lifetime values for fluorochromes with emission peaks in the visible or near infrared
<p>Multiplexing techniques rely on fluorescent probes to simultaneously detect and visualise multiple mRNA or protein molecules in a single cell. Although more than 1200 fluorochromes are available in the visible and near-infrared spectral range, it is difficult to separate the different fluorochromes spectrally into orthogonal channels as their excitation and emission spectra often overlap. Fluorescence lifetimes can be used as an effective method to segregate fluorochromes for multiplex imaging. However, information on fluorescence lifetimes is not always easy to find as it is often only mentioned in passing on websites or in publications. In an effort to overcome this challenge, we performed a systematic literature review to make it easier to access the information required to attempt unmixing fluorochromes by fluorescence lifetime for multiplexed imaging. We found that at least 88 fluorochromes can be used, in principle, to attempt unmixing fluorochromes by lifetime and thus multiplexing. Our data are summarised in a table as well as in a graph in which we plotted the lifetime (tau) against the emission peak (Em). For all fluorochromes, we found that are available as NHS derivatives for easy coupling to DNA oligonucleotides or antibodies. Some additional, potentially very useful fluorophores, for which no lifetime data are available, are shown within a dashed line in a relatively “sparse” region of the spectrum above 800nm. A key is displayed to describe the colour code used to represent different fluorochrome classes, such as Alexa Fluor.</p>
Data for "Nanoparticle doping and molten-core methods towards highly thulium-doped silica fibers for 0.79 μm-pumped 2 μm fiber lasers – a fluorescence lifetime study"
<p>Includes data for basic characterization of the fibers (concentration profiles from EMPA, refractive index profiles of the preforms and fibers, attenuation of the fibers), as well as the measured fluorescence decay curves.</p>
Real‐time fiber‐based fluorescence lifetime imaging with synchronous external illumination: A new path for clinical translation
<p>Time-correlated single photon counting is the “gold-standard” method for fluorescence lifetime measurements and has demonstrated potential for clinical deployment. Its clinical adoption is hindered by the use of high gain detectors, which make the fluorescence acquisition impractical with bright lighting conditions such as in clinical settings. We address this limitation by interleaving periodic fluorescence detection with synchronous out-of-phase externally modulated light source, thus guaranteeing specimen illumination and a fluorescence signal free from bright background light upon temporal separation. Fluorescence lifetime maps are generated in real-time from single-point measurements by tracking a reference beam and using the phasor approach. We demonstrate the feasibility and practicality of this technique in a number of biological specimens, including real-time mapping of degraded articular cartilage. This method is compatible and can be integrated with existing clinical microscopic, endoscopic and robotic modalities, thus offering a new pathway towards label-free diagnostics and surgical guidance in a number of clinical applications.</p>
Wide-field fluorescence lifetime imaging of neuron spiking and sub-threshold activity in vivo
Open the record for dataset details and reuse information.
Data from the paper "A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium"
<p>Data that belongs to the paper "A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium"</p> <p> </p>
Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin
<p>Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.</p>
Measurement of Retinal Auto Fluorescence With a Fluorescence Lifetime Imaging Ophthalmoscope
ClinicalTrials.gov study NCT01981148. IPD Sharing: Not stated. Countries: 1. Publications: 8.
The Effect of Hyperoxia and Hypoxia on Fluorescence Lifetime Imaging Ophthalmoscopy in Healthy Subjects- a Randomized, Double Blind, Crossover Study
ClinicalTrials.gov study NCT04094285. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Data repository for the article: Fluorescence lifetime imaging unravels the pathway of glioma cell death upon hypericin-induced photodynamic therapy.
Open the record for dataset details and reuse information.
Structural Imaging Assisted Retinal Fluorescence Lifetime Imager (STARFLI)
ClinicalTrials.gov study NCT05819307. IPD Sharing: NO. Countries: 1. Publications: 0.
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