Spectrally resolved fluorescent lifetime imaging

Hanley, Quentin S.

doi:10.1098/rsif.2008.0393.focus

Cited by 18 publications

(13 citation statements)

References 47 publications

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“…This similar straight line behaviour of three lifetimes has also been shown in a simulated polar plot diagram in a review by Hanley (2009); however, no connection with the spectrum was made there. However, there is a mislabelling in Fig.…”

Section: Methodssupporting

confidence: 77%

“…From Eq. (9a), the acceptor modulation M A e and phase ϕ A e can be expressed as (Birks, 1968, 1970; Lakowicz & Balter, 1982; Chen et al , 2009b; Hanley, 2009): where the subscript e denotes that the fluorescence signal of the acceptor is excited by energy transfer. shows that the acceptor modulation with FRET equals the multiplication of the acceptor modulation without FRET (direct excitation) and the donor modulation with FRET.…”

Section: Methodsmentioning

confidence: 99%

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Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

Chen

Clegg

2011

Journal of Microscopy

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SummaryA spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetimeresolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime -and thereby the FRET efficiency -without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.

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Section: Methodssupporting

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

Chen

Clegg

2011

Journal of Microscopy

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“…Advances in integrated SPAD sensor arrays promise much faster acquisition of fluorescence lifetime data in live cell imaging [14]. Spectroscopy is particularly attractive as time-resolved emission (fluorescence) spectroscopy (TRES) contains multi-dimensional information across temporal and spectral axes which can be used to derive spectral relaxation properties and fluorescence lifetimes, see chapter 7 in [1] and [15,16]. TRES has a long history and the first systems were implemented in 1970's, for example see [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy

Krstajić¹,

Levitt²,

Poland³

et al. 2015

Opt. Express

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We present a CMOS chip 256 × 2 single photon avalanche diode (SPAD) line sensor, 23.78 µm pitch, 43.7% fill factor, custom designed for time resolved emission spectroscopy (TRES). Integrating time-to-digital converters (TDCs) implement on-chip mono-exponential fluorescence lifetime pre-calculation allowing timing of 65k photons/pixel at 200 Hz line rate at 40 ps resolution using centre-of-mass method (CMM). Per pixel time-correlated single-photon counting (TCSPC) histograms can also be generated with 320 ps bin resolution. We characterize performance in terms of dark count rate, instrument response function and lifetime uniformity for a set of fluorophores with lifetimes ranging from 4 ns to 6 ns. Lastly, we present fluorescence lifetime spectra of multicolor microspheres and skin autofluorescence acquired using a custom built spectrometer. In TCSPC mode, time-resolved spectra are acquired within 5 minutes whilst in CMM mode spectral lifetime signatures are acquired within 2 ms for fluorophore in cuvette and 200 ms for skin autofluorescence. We demonstrate CMOS line sensors to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay.

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“…Such a resolution of acceptor emission from direct excitation and via FRET is discussed in details elsewhere. 29 Raw FLIM data for the FRET system of TLR4-Cer/4BB-BTX are shown in Fig. 4(a).…”

Section: Background Correction Of Flim Datamentioning

confidence: 99%

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

2014

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Abstract. Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.

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Spectrally resolved fluorescent lifetime imaging

Cited by 18 publications

References 47 publications

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

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