Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

Trinel, Dave; Leray, Aymeric; Spriet, Corentin; Usson, Yves; Héliot, Laurent

doi:10.1002/cyto.a.21054

Cited by 14 publications

(16 citation statements)

References 32 publications

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“…More details on the algorithm can be found in [27], [28]. In order to be as close as possible of the experimental conditions, we consider a laser repetition frequency of 80 MHz which corresponds to a total window width of 12.5 ns.…”

Section: Methodsmentioning

confidence: 99%

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

et al. 2013

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Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mfD. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels Nch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, Nch or small fractions of interacting donor fD). The validity domain of the moments method is more restricted (not applicable when fD<0.25 or when Nch = 4) but it is more precise than the polar approach. We also demonstrate that the mfD is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.

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Section: Methodsmentioning

confidence: 99%

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

et al. 2013

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“…Improvement of the signal-to-noise ratio indicates that CytD allows analyzing FRET also when the tagged partners occur in small quantities. This is also relevant when FRET is studied using intensitybased, ratiometric approaches (19,37), a technique that can easily be implemented in wide field and confocal microscopes, as well as for fluorescent lifetime imaging microscopy (20).…”

Section: Rough Estimation Of Fret Based On Spectral Imagesmentioning

confidence: 99%

“…To study the interaction of proteins in vivo, tagging with fluorescent proteins is the method of choice (14,15). FRET between CFP and YFP can be measured and analyzed in a broad range of microscopic modalities (16,17), including the simple approach of acceptor photobleaching (18), the more involved intensitybased ratiometric approach (19), or the technically quite challenging lifetime-based approaches (17,20).…”

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confidence: 99%

Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein‐tagged Golgi components

et al. 2013

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Protein-protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Förster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein-protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton. ' International Society for Advancement of Cytometry Key termsCytochalasin D; cytoplasmic streaming; fluorescent proteins; FRET; Golgi; glycosyltransferase; fixation; Nicotiana benthamiana; paraformaldehyde; plants; spectral imaging FOR protein-protein interaction studies in live cells, spatial changes over time such as cytoplasmic streaming are a great challenge. Cytoplasmic streaming is characteristic for intracellular transport in large cells and accompanies cell motility. It leads to the displacements of organelles and RNA processing stacks, mixing of the cytosol and reinforcement of cell polarity (1,2). As a consequence, proteins of interest rapidly move through the observed cellular region. The velocity of organelle transport can reach 10 lm s 21 ; for Golgi stacks and vesicles, values between 4.2 and 7 lm s 21 have been documented in plant cells and on average 2.1 lm s 21 in neuronal axons, respectively (3-5). In plants, cytoplasmic streaming involves the cytoskeletal actin-filaments and myosin motor proteins, as opposed to animal cells, where microtubules and kinesin or dynein are used (6-8).The Golgi apparatus of plants is central for glycosylation and secretory processes including synthesis of cell wall polysaccharides during cell differentiation. The importance

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“…In real experiments, the detected intensity pro¯le IðtÞ is the convolution product of F ðtÞ, the°uorescence background and the IRF of the TCSPC-FLIM system, and thus can be described by 14,17 IðtÞ ¼ ðF ðtÞ þ BackgroundÞ IRF:…”

Section: Introductionmentioning

confidence: 99%

Quantifying the Short Lifetime With TCSPC-Flim: First Moment Versus Fitting Methods

Wei

Zeng

et al. 2013

J. Innov. Opt. Health Sci.

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Combing the time-correlated single photon counting (TCSPC) with°uorescence lifetime imaging microscopy (FLIM) provides promising opportunities in revealing important information on the microenvironment of cells and tissues, but the applications are thus far mainly limited by the accuracy and precision of the TCSPC-FLIM technique. Here we present a comprehensive investigation on the performance of two data analysis methods, the¯rst moment (M 1 ) method and the conventional least squares (Fitting) method, in quantifying°uorescence lifetime. We found that the M 1 method is more superior than the Fitting method when the lifetime is short (70 $ 400 ps) or the signal intensity is weak (< 10 3 photons).

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Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

Cited by 14 publications

References 32 publications

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein‐tagged Golgi components

Quantifying the Short Lifetime With TCSPC-Flim: First Moment Versus Fitting Methods

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