Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.

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KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

European Biophysics Journal ◽

10.1007/s00249-021-01560-2 ◽

2021 ◽

Author(s):

Diana Spiegelberg ◽

Jonas Stenberg ◽

Pascale Richalet ◽

Marc Vanhove

Keyword(s):

Flow Cytometry ◽

Live Cells ◽

Time Resolved ◽

Surface Receptors ◽

Full Characterization ◽

End Point ◽

Titration Curves ◽

Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

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Thulium-Doped Silica Fibers with Enhanced Fluorescence Lifetime and Their Application in Ultrafast Fiber Lasers

Fibers ◽

10.3390/fib6030066 ◽

2018 ◽

Vol 6 (3) ◽

pp. 66 ◽

Cited By ~ 12

Author(s):

Jakub Cajzl ◽

Pavel Peterka ◽

Maciej Kowalczyk ◽

Jan Tarka ◽

Grzegorz Sobon ◽

...

Keyword(s):

Optical Fibers ◽

Fluorescence Lifetime ◽

Energy Levels ◽

Local Environment ◽

Alumina Nanoparticles ◽

Fluorescence Lifetimes ◽

Transfer Coefficients ◽

Relaxation Energy ◽

Time Resolved ◽

Conventional Solution

In this work we report on the thulium-doped silica-based optical fibers with increased fluorescence lifetime of the 3F4 level thanks to the modification of the local environment of thulium ions by high content of alumina. The determination of the cross-relaxation energy-transfer coefficients from the measurements of the fluorescence lifetimes of the 3F4 and 3H4 energy levels of Tm3+ ions in the experimentally prepared optical fiber is provided as well. Preforms of optical fibers were prepared either by conventional solution-doping of Tm3+ and Al3+ ions or by dispersion-doping of Tm3+ ions with alumina nanoparticles. Optical fibers were characterized by means of Tm, Al, and Ge concentrations, refractive index profiles, optical spectral absorption and luminescence, and by time-resolved fluorescence spectroscopy. Highly aluminium-codoped thulium silicate optical fibers exhibited fluorescence lifetimes of over ~500 μs with maximum value of 756 μs, which means a fluorescence lifetime enhancement when compared to the thulium-doped fibers reported elsewhere. We show an application of the thulium-doped fiber in a compact all-fiber ring laser that is passively mode-locked by using graphene-based saturable absorber. The output pulsewidth and repetition rate were 905 fs and 32.67 MHz, respectively.

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The biochemical resolving power of fluorescence lifetime imaging

10.1101/2021.02.03.429635 ◽

2021 ◽

Author(s):

Andrew L. Trinh ◽

Alessandro Esposito

Keyword(s):

Fluorescence Lifetime ◽

Resolving Power ◽

Fluorescence Lifetime Imaging ◽

Time Resolved ◽

Instrument Response Function ◽

Lifetime Imaging ◽

Use Of Time ◽

Single Living Cells ◽

Microscopy Techniques

AbstractA deeper understanding of spatial resolution in microscopy fostered a technological revolution that is now permitting us to investigate the structure of the cell with nanometer resolution. Although fluorescence microscopy techniques enable scientists to investigate both the structure and biochemistry of the cell, the biochemical resolving power of a microscope is a physical quantity that is not well-defined or studied. To overcome this limitation, we carried out a theoretical investigation of the biochemical resolving power in fluorescence lifetime imaging microscopy, one of the most effective tools to investigate biochemistry in single living cells. With the theoretical analysis of information theory and Monte Carlo simulations, we describe how the ‘biochemical resolving power’ in time-resolved sensing depends on instrument specifications. We unravel common misunderstandings on the role of the instrument response function and provide theoretical insights that have significant practical implications in the design and use of time-resolved instrumentation.

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Quantifying cell densities and biovolumes of phytoplankton communities and functional groups using scanning flow cytometry, machine learning and unsupervised clustering

10.1101/274357 ◽

2018 ◽

Author(s):

Mridul K. Thomas ◽

Simone Fontana ◽

Marta Reyes ◽

Francesco Pomati

Keyword(s):

Machine Learning ◽

Flow Cytometry ◽

Functional Groups ◽

Clustering Algorithm ◽

Unsupervised Clustering ◽

Learning Tools ◽

Time Resolved ◽

Automated Method ◽

Phytoplankton Communities ◽

Cell Densities

AbstractScanning flow cytometry (SFCM) is characterized by the measurement of time-resolved pulses of fluorescence and scattering, enabling the high-throughput quantification of phytoplankton morphology and pigmentation. Quantifying variation at the single cell and colony level improves our ability to understand dynamics in natural communities. Automated high-frequency monitoring of these communities is presently limited by the absence of repeatable, rapid protocols to analyse SFCM datasets, where images of individual particles are not available. Here we demonstrate a repeatable, semi-automated method to (1) rapidly clean SFCM data from a phytoplankton community by removing signals that do not belong to live phytoplankton cells, (2) classify individual cells into trait clusters that correspond to functional groups, and (3) quantify the biovolumes of individual cells, the total biovolume of the whole community and the total biovolumes of the major functional groups. Our method involves the development of training datasets using lab cultures, the use of an unsupervised clustering algorithm to identify trait clusters, and machine learning tools (random forests) to (1) evaluate variable importance, (2) classify data points, and (3) estimate biovolumes of individual cells. We provide example datasets and R code for our analytical approach that can be adapted for analysis of datasets from other flow cytometers or scanning flow cytometers.

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Progress toward combined phase and amplitude demodulation fluorescence lifetime measurements by flow cytometry

10.1117/12.307098 ◽

1998 ◽

Cited By ~ 1

Author(s):

John A. Steinkamp ◽

Jimmie D. Parson ◽

Jan F. Keij

Keyword(s):

Flow Cytometry ◽

Fluorescence Lifetime ◽

Lifetime Measurements

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Fluorescence lifetime-based sensing of polymersome leakage

Photochemical & Photobiological Sciences ◽

10.1039/c6pp00296j ◽

2017 ◽

Vol 16 (2) ◽

pp. 155-158 ◽

Cited By ~ 2

Author(s):

Stephan Wang ◽

Zhong-Ren Chen

Keyword(s):

Fluorescence Lifetime ◽

Polymer Degradation ◽

Fluorescence Technique ◽

Fluorescence Lifetimes ◽

Time Resolved Fluorescence ◽

Time Resolved

The time-resolved fluorescence technique enables us to differentiate between polymer degradation and vesicle leakage by employing fluorescence lifetimes and their amplitudes.

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