The Phasor Approach to Fluorescence Lifetime Imaging Analysis

Digman, Michelle A.; Caiolfa, Valeria R.; Zamai, Moreno; Gratton, Enrico

doi:10.1529/biophysj.107.120154

Cited by 1,004 publications

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“…46 The fluorescence lifetimes of unbound DOX and P(MAA-co-CMA)-DOX nanocomplexes in aqueous solution were measured via time-correlated single photon counting (TCSPC) and represented in a phasor plot. 47,48 The lifetime of each species is categorized in pixel format and transformed into a phasor plot by fitting the data to an exponential decay. All single exponential lifetimes lie on the universal circle while multi-exponential lifetimes are a linear combination of their components (Scheme S2, ESI †).…”

Section: Characterization Studies Of P(maa-co-cma)-dox Nanocomplexesmentioning

confidence: 99%

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

et al. 2015

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Previously synthesized poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA) copolymers were examined as potential drug delivery vehicles. P(MAA-co-CMA) copolymers were fluorescently labelled and imaged in SHEP and HepG2 cells. To understand their cell internalization pathway endocytic inhibition studies were conducted. It was concluded that P(MAA-co-CMA) are taken up by the cells via clathrin-independent endocytosis (CIE) (both caveolae mediated and cholesterol dependent endocytosis) mechanisms. The formation and characterization of P(MAA-co-CMA)-doxorubicin (DOX) nanocomplexes was investigated by fluorescence lifetime imaging microscopy (FLIM), UV-Visible spectroscopy (UV-Vis) and dynamic light scattering (DLS) studies. The toxicity screening between P(MAA-co-CMA)-DOX nanocomplexes (at varying w/w ratios) and free DOX, revealed nanocomplexes to exhibit higher cytotoxicity towards cancer cells in comparison to normal cells. FLIM and confocal microscopy were employed for investigating the time-dependent release of DOX in SHEP cells and the cellular uptake profile of P(MAAco-CMA)-DOX nanocomplexes in cancer and normal cell lines, respectively. The endocytic pathway of P(MAA-co-CMA)-DOX nanocomplexes were examined in SHEP and HepG2 cells via flow cytometry revealing the complexes to be internalized through both clathrin-dependent (CDE) and CIE mechanisms. The drug delivery profile, reported herein, illuminates the specific endocytic route and therapeutic efficiency of P(MAA-co-CMA)-DOX nanocomplexes strongly suggesting these particles to be promising candidates for in vivo applications.

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Section: Characterization Studies Of P(maa-co-cma)-dox Nanocomplexesmentioning

confidence: 99%

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

et al. 2015

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“…In this article, we propose to extend this approach to SLIM data acquired in the temporal domain with time correlated single photon counting (TCSPC) technique. Polar calculations for each spectral interval were based on the previous work realized by E. Gratton and coworkers (16). Technically, for each pixel, the experimental results gathered with TCSPC technique are converted into frequency-domain data with a simple Fourier transform of the temporal decay I(t) and this complex value is separated into real (Re) and imaginary (Im) parts to obtain the [u; v] coordinates in the polar representation, also called an Argand diagram (Fig.…”

Section: Polar Representationmentioning

confidence: 99%

“…The case of a mixture of several molecular species and the case of FRET have already been studied (16,19). …”

Section: Polar Representationmentioning

confidence: 99%

“…Recently, many efforts have been made to help simplify the results interpretation in classic FLIM experiments for nonspecialists (15)(16)(17). Among all these techniques, the polar plot or phasor (16,(18)(19)(20) is a promising approach that avoids complex fitting algorithm strategies and provides a fast, graphical representation of fluorescence lifetimes.…”

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confidence: 99%

“…Among all these techniques, the polar plot or phasor (16,(18)(19)(20) is a promising approach that avoids complex fitting algorithm strategies and provides a fast, graphical representation of fluorescence lifetimes. This polar approach has successfully been applied in classic FLIM and FRET experiments (single spectral channel) by Digman et al (16).…”

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Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

et al. 2009

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Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent. ' 2009 International Society for Advancement of Cytometry Key terms multispectral FLIM; SLIM; biological tissue; molecular interactions; FRET; phasor IN addition to intensity and wavelength, lifetime is a supplementary source of contrast in fluorescence microscopy, which greatly increases access to the biological information in living cells. Fluorescence Lifetime Imaging Microscopy (FLIM) has indeed been successfully applied to differentiate spectrally undistinguishable molecular species (1) and to map local modifications in labeled samples in terms of ion concentration (2), pH (3), and oxygen (4). FLIM has also been widely used to explore conformational change of proteins (5) or to separate interacting and non-interacting molecular fractions in Förster Resonance Energy Transfer (FRET) experiments (6,7).However, because of the low number of collected photons per pixel and the variability of the molecular environment inside a living cell, FLIM is usually limited to two lifetime components in the same pixel. This restriction makes it difficult to extract biologically relevant information from autofluorescent, heterogeneous, lightscattering samples such as biological tissue. Because of optical properties of tissue, a dedicated temporally and spectrally resolved system (called SLIM for Spectral and Lifetime Imaging Microscopy) is necessary for example to be able to explore molecular interactions on a nanometric scale (with FRET experiments).Temporal and spectral measurements can be performed using two approaches, either frequency domain (8,9) or time domain (10-12) measurements. In both cases, the determination of lifetimes and contributions of each molecular species are mainly achieved by fitting the collected data at each pixel using equations with multiple

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Fluorescence Microscopy

Valeur

Berberan‐Santos²

2012

Molecular Fluorescence

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The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in dwindow chambersT in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function. D

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The Phasor Approach to Fluorescence Lifetime Imaging Analysis

Cited by 1,004 publications

References 14 publications

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

Fluorescence Microscopy

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